Combinatorial DNA library for producing modified N-glycans in lower eukaryotes

ABSTRACT

The present invention relates to eukaryotic host cells having modified oligosaccharides which may be modified further by heterologous expression of a set of glycosyltransferases, sugar transporters and mannosidases to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. The invention provides nucleic acid molecules and combinatorial libraries which can be used to successfully target and express mammalian enzymatic activities such as those involved in glycosylation to intracellular compartments in a eukaryotic host cell. The process provides an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation. Host cells with modified oligosaccharides are created or selected. N-glycans made in the engineered host cells have a Man 5 GlcNAc 2  core structure which may then be modified further by heterologous expression of one or more enzymes, e.g., glycosyltransferases, sugar transporters and mannosidases, to yield human-like glycoproteins. For the production of therapeutic proteins, this method may be adapted to engineer cell lines in which any desired glycosylation structure may be obtained.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/892,591, filed Jun. 27, 2001, in which priority is claimedto U.S. Provisional Application Serial No. 60/214,358, filed Jun. 28,2000, U.S. Provisional Application No. 60/215,638, filed Jun. 30, 2000,and U.S. Provisional Application No. 60/279,997, filed Mar. 30, 2001;each of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention is directed to methods and compositions bywhich non-human eukaryotic host cells, such as fungi or other eukaryoticcells, can be genetically modified to produce glycosylated proteins(glycoproteins) having patterns of glycosylation similar to those ofglycoproteins produced by animal cells, especially human cells, whichare useful as human or animal therapeutic agents.

BACKGROUND OF THE INVENTION Glycosylation Pathways in Humans and LowerEukaryotes

[0003] After DNA is transcribed and translated into a protein, furtherpost-translational processing involves the attachment of sugar residues,a process known as glycosylation. Different organisms produce differentglycosylation enzymes (glycosyltransferases and glycosidases), and havedifferent substrates (nucleotide sugars) available, so that theglycosylation patterns as well as composition of the individualoligosaccharides, even of the same protein, will be different dependingon the host system in which the particular protein is being expressed.Bacteria typically do not glycosylate proteins, and if so only in a veryunspecific manner (Moens and Vanderleyden, 1997 Arch Microbiol.168(3):169-175). Lower eukaryotes such as filamentous fungi and yeastadd primarily mannose and mannosylphosphate sugars. The resulting glycanis known as a “high-mannose” type glycan or a mannan. Plant cells andinsect cells (such as Sf9 cells) glycosylate proteins in yet anotherway. By contrast, in higher eukaryotes such as humans, the nascentoligosaccharide side chain may be trimmed to remove several mannoseresidues and elongated with additional sugar residues that typically arenot found in the N-glycans of lower eukaryotes. See, e.g., R. K.Bretthauer, et al. Biotechnology and Applied Biochemistry, 1999, 30,193-200; W. Martinet, et al. Biotechnology Letters, 1998, 20, 1171-1177;S. Weikert, et al. Nature Biotechnology, 1999, 17, 1116-1121; M.Malissard, et al. Biochemical and Biophysical Research Communications,2000, 267, 169-173; Jarvis, et al., Current Opinion in Biotechnology,1998, 9:528-533; and M. Takeuchi, 1 Trends in Glycoscience andGlycotechnology, 1997, 9, S29-S35.

[0004] Synthesis of a mammalian-type oligosaccharide structure beginswith a set of sequential reactions in the course of which sugar residuesare added and removed while the protein moves along the secretorypathway in the host organism. The enzymes which reside along theglycosylation pathway of the host organism or cell determine theresulting glycosylation patterns of secreted proteins. Thus, theresulting glycosylation pattern of proteins expressed in lowereukaryotic host cells differs substantially from the glycosylationpattern of proteins expressed in higher eukaryotes such as humans andother mammals (Bretthauer, 1999). The structure of a typical fungalN-glycan is shown in FIG. 1A.

[0005] The early steps of human glycosylation can be divided into atleast two different phases: (i) lipid-linked Glc₃Man₉GlcNAc₂oligosaccharides are assembled by a sequential set of reactions at themembrane of the endoplasmic reticulum (ER) and (ii) the transfer of thisoligosaccharide from the lipid anchor dolichyl pyrophosphate onto denovo synthesized protein. The site of the specific transfer is definedby an asparagine (Asn) residue in the sequence Asn-Xaa-Ser/Thr where Xaacan be any amino acid except proline (Gavel, 1990). Further processingby glucosidases and mannosidases occurs in the ER before the nascentglycoprotein is transferred to the early Golgi apparatus, whereadditional mannose residues are removed by Golgi specific alpha(α)-1,2-mannosidases. Processing continues as the protein proceedsthrough the Golgi. In the medial Golgi, a number of modifying enzymes,including N-acetylglucosaminyl Transferases (GnTI, GnTII, GnTIII, GnTIVand GnTV), mannosidase II and fucosyltransferases, add and removespecific sugar residues. Finally, in the trans-Golgi,galactosyltranferases (GalT) and sialyltransferases (ST) produce aglycoprotein structure that is released from the Golgi. It is thisstructure, characterized by bi-, tri- and tetra-antennary structures,containing galactose, fucose, N-acetylglucosamine and a high degree ofterminal sialic acid, that gives glycoproteins their humancharacteristics. The structure of a typical human N-glycan is shown inFIG. 1B.

[0006] In nearly all eukaryotes, glycoproteins are derived from a commonlipid-linked oligosaccharide precursorGlc₃Man₉GlcNAc₂-dolichol-pyrophosphate. Within the endoplasmicreticulum, synthesis and processing of dolichol pyrophosphate boundoligosaccharides are identical between all known eukaryotes. However,further processing of the core oligosaccharide by fungal cells, e.g.,yeast, once it has been transferred to a peptide leaving the ER andentering the Golgi, differs significantly from humans as it moves alongthe secretory pathway and involves the addition of several mannosesugars.

[0007] In yeast, these steps are catalyzed by Golgi residingmannosyltransferases, like Och1p, Mnt1p and Mnn1p, which sequentiallyadd mannose sugars to the core oligosaccharide. The resulting structureis undesirable for the production of human-like proteins and it is thusdesirable to reduce or eliminate mannosyltransferase activity. Mutantsof S. cerevisiae, deficient in mannosyltransferase activity (for exampleoch1 or mnn9 mutants) have been shown to be non-lethal and displayreduced mannose content in the oligosaccharide of yeast glycoproteins.Other oligosaccharide processing enzymes, such as mannosylphosphatetransferase, may also have to be eliminated depending on the host'sparticular endogenous glycosylation pattern.

Sugar Nucleotide Precursors

[0008] The N-glycans of animal glycoproteins typically includegalactose, fucose, and terminal sialic acid. These sugars are not foundon glycoproteins produced in yeast and filamentous fungi. In humans, thefull range of nucleotide sugar precursors (e.g. UDP-N-acetylglucosamine,UDP-N-acetylgalactosamine, CMP-N-acetylneuraminic acid, UDP-galactose,GDP-fucose, etc.) are synthesized in the cytosol and transported intothe Golgi, where they are attached to the core oligosaccharide byglycosyltransferases. (Sommers and Hirschberg, 1981 J. Cell Biol. 91(2):A406-A406; Sommers and Hirschberg 1982 J. Biol. Chem. 257(18): 811-817;Perez and Hirschberg 1987 Methods in Enzymology 138: 709-715).

[0009] Glycosyl transfer reactions typically yield a side product whichis a nucleoside diphosphate or monophosphate. While monophosphates canbe directly exported in exchange for nucleoside triphosphate sugars byan antiport mechanism, diphosphonucleosides (e.g. GDP) have to becleaved by phosphatases (e.g. GDPase) to yield nucleoside monophosphatesand inorganic phosphate prior to being exported. This reaction isimportant for efficient glycosylation; for example, GDPase fromSaccharomyces cerevisiae (S. cerevisiae) has been found to be necessaryfor mannosylation. However that GDPase has 90% reduced activity towardUDP (Berninsone et al., 1994 J. Biol. Chem. 269(1):207-211). Lowereukaryotes typically lack UDP-specific diphosphatase activity in theGolgi since they do not utilize UDP-sugar precursors for Golgi-basedglycoprotein synthesis. Schizosaccharomyces pombe, a yeast found to addgalactose residues to cell wall polysaccharides (from UDP-galactose) hasbeen found to have specific UDPase activity, indicating the potentialrequirement for such an enzyme (Berninsone et al., 1994). UDP is knownto be a potent inhibitor of glycosyltransferases and the removal of thisglycosylation side product may be important to preventglycosyltransferase inhibition in the lumen of the Golgi (Khatara etal., 1974). See Berninsone, P., et al. 1995. J. Biol. Chem. 270(24):14564-14567; Beaudet, L., et al. 1998 Abc Transporters: Biochemical,Cellular, and Molecular Aspects. 292: 397-413.

Sequential Processing of N-glycans by Compartmentalized EnzymeActivities

[0010] Sugar transferases and glycosidases (e.g., mannosidases) line theinner (luminal) surface of the ER and Golgi apparatus and therebyprovide a “catalytic” surface that allows for the sequential processingof glycoproteins as they proceed through the ER and Golgi network. Themultiple compartments of the cis, medial, and trans Golgi and thetrans-Golgi Network (TGN), provide the different localities in which theordered sequence of glycosylation reactions can take place. As aglycoprotein proceeds from synthesis in the ER to full maturation in thelate Golgi or TGN, it is sequentially exposed to different glycosidases,mannosidases and glycosyltransferases such that a specific carbohydratestructure may be synthesized. Much work has been dedicated to revealingthe exact mechanism by which these enzymes are retained and anchored totheir respective organelle. The evolving picture is complex but evidencesuggests that stem region, membrane spanning region and cytoplasmictail, individually or in concert, direct enzymes to the membrane ofindividual organelles and thereby localize the associated catalyticdomain to that locus (see, e.g., Gleeson, P. A. (1998) Histochem. CellBiol. 109, 517-532).

[0011] In some cases, these specific interactions were found to functionacross species. For example, the membrane spanning domain of α2,6-STfrom rats, an enzyme known to localize in the trans-Golgi of the animal,was shown to also localize a reporter gene (invertase) in the yeastGolgi (Schwientek, 1995). However, the very same membrane spanningdomain as part of a full-length α2,6-ST was retained in the ER and notfurther transported to the Golgi of yeast (Krezdorn, 1994). A fulllength GalT from humans was not even synthesized in yeast, despitedemonstrably high transcription levels. In contrast, the transmembraneregion of the same human GalT fused to an invertase reporter was able todirect localization to the yeast Golgi, albeit it at low productionlevels. Schwientek and co-workers have shown that fusing 28 amino acidsof a yeast mannosyltransferase (MNT1), a region containing a cytoplasmictail, a transmembrane region and eight amino acids of the stem region,to the catalytic domain of human GalT are sufficient for Golgilocalization of an active GalT. Other galactosyltransferases appear torely on interactions with enzymes resident in particular organellesbecause, after removal of their transmembrane region, they are stillable to localize properly.

[0012] Improper localization of a glycosylation enzyme may preventproper functioning of the enzyme in the pathway. For example,Aspergillus nidulans, which has numerous α-1,2-mannosidases (Eades andHintz, 2000 Gene 255(1):25-34), does not add GlcNAc to Man₅GlcNAc₂ whentransformed with the rabbit GnTI gene, despite a high overall level ofGnTI activity (Kalsner et al., 1995). GnTI, although actively expressed,may be incorrectly localized such that the enzyme is not in contact withboth of its substrates: UDP-GlcNAc and a productive Man₅GlcNAc₂substrate (not all Man₅GlcNAc₂ structures are productive; see below).Alternatively, the host organism may not provide an adequate level ofUDP-GlcNAc in the Golgi or the enzyme may be properly localized butnevertheless inactive in its new environment. In addition, Man₅GlcNAc₂structures present in the host cell may differ in structure fromMan₅GlcNAc₂ found in mammals. Maras and coworkers found that about onethird of the N-glycans from cellobiohydrolase I (CBHI) obtained from T.reesei can be trimmed to Man₅GlcNAc₂ by A.saitoi 1,2 mannosidase invitro. Fewer than 1% of those N-glycans, however, could serve as aproductive substrate for GnTI. The mere presence of Man₅GlcNAc₂,therefore, does not assure that further processing to Man₅GlcNAc₂ can beachieved. It is formation of a productive, GnTI-reactive Man₅GlcNAc₂structure that is required. Although Man₅GlcNAc₂ could be produced inthe cell (about 27 mol %), only a small fraction could be converted toMan₅GlcNAc₂ (less than about 5%, see Chiba WO 01/14522).

[0013] To date, there is no reliable way of predicting whether aparticular heterologously expressed glycosyltransferase or mannosidasein a lower eukaryote will be (1), sufficiently translated (2),catalytically active or (3) located to the proper organelle within thesecretory pathway. Because all three of these are necessary to affectglycosylation patterns in lower eukaryotes, a systematic scheme toachieve the desired catalytic function and proper retention of enzymesin the absence of predictive tools, which are currently not available,would be desirable.

Production of Therapeutic Glycoproteins

[0014] A significant number of proteins isolated from humans or animalsare post-translationally modified, with glycosylation being one of themost significant modifications. An estimated 70% of all therapeuticproteins are glycosylated and thus currently rely on a production system(i.e., host cell) that is able to glycosylate in a manner similar tohumans. Several studies have shown that glycosylation plays an importantrole in determining the (1) immunogenicity, (2) pharmacokineticproperties, (3) trafficking, and (4) efficacy of therapeutic proteins.It is thus not surprising that substantial efforts by the pharmaceuticalindustry have been directed at developing processes to obtainglycoproteins that are as “humanoid” or “human-like” as possible. Todate, most glycoproteins are made in a mammalian host system. This mayinvolve the genetic engineering of such mammalian cells to enhance thedegree of sialylation (i.e., terminal addition of sialic acid) ofproteins expressed by the cells, which is known to improvepharmacokinetic properties of such proteins. Alternatively, one mayimprove the degree of sialylation by in vitro addition of such sugarsusing known glycosyltransferases and their respective nucleotide sugars(e.g., 2,3-sialyltransferase and CMP-sialic acid).

[0015] While most higher eukaryotes carry out glycosylation reactionsthat are similar to those found in humans, recombinant human proteinsexpressed in the above mentioned host systems invariably differ fromtheir “natural” human counterpart (Raju, 2000). Extensive developmentwork has thus been directed at finding ways to improve the “humancharacter” of proteins made in these expression systems. This includesthe optimization of fermentation conditions and the genetic modificationof protein expression hosts by introducing genes encoding enzymesinvolved in the formation of human-like glycoforms (Werner, 1998;Weikert, 1999; Andersen, 1994; Yang, 2000). Inherent problems associatedwith all mammalian expression systems have not been solved.

[0016] Fermentation processes based on mammalian cell culture (e.g.,CHO, murine, or human cells), for example, tend to be very slow(fermentation times in excess of one week are not uncommon), often yieldlow product titers, require expensive nutrients and cofactors (e.g.,bovine fetal serum), are limited by programmed cell death (apoptosis),and often do not enable expression of particular therapeuticallyvaluable proteins. More importantly, mammalian cells are susceptible toviruses that have the potential to be human pathogens and stringentquality controls are required to assure product safety. This is ofparticular concern as many such processes require the addition ofcomplex and temperature sensitive media components that are derived fromanimals (e.g., bovine calf serum), which may carry agents pathogenic tohumans such as bovine spongiform encephalopathy (BSE) prions or viruses.Moreover, the production of therapeutic compounds is preferably carriedout in a well-controlled sterile environment. An animal farm, no matterhow cleanly kept, does not constitute such an environment, thusconstituting an additional problem in the use of transgenic animals formanufacturing high volume therapeutic proteins.

[0017] Most, if not all, currently produced therapeutic glycoproteinsare therefore expressed in mammalian cells and much effort has beendirected at improving (i.e., “humanizing”) the glycosylation pattern ofthese recombinant proteins. Changes in medium composition as well as theco-expression of genes encoding enzymes involved in human glycosylationhave been successfully employed (see, for example, Weikert, 1999).

Glycoprotein Production Using Eukaryotic Microorganisms

[0018] The lack of a suitable mammalian expression system is asignificant obstacle to the low-cost and safe production of recombinanthuman glycoproteins for therapeutic applications. It would be desirableto produce recombinant proteins similar to their mammalian, e.g., human,counterparts in lower eukaryotes (fungi and yeast). Production ofglycoproteins via the fermentation of microorganisms would offernumerous advantages over existing systems. For example,fermentation-based processes may offer (a) rapid production of highconcentrations of protein; (b) the ability to use sterile,well-controlled production conditions; (c) the ability to use simple,chemically defined (and protein-free) growth media; (d) ease of geneticmanipulation; (e) the absence of contaminating human or animal pathogenssuch as viruses; (f) the ability to express a wide variety of proteins,including those poorly expressed in cell culture owing to toxicity etc.;and (g) ease of protein recovery (e.g. via secretion into the medium).In addition, fermentation facilities for yeast and fungi are generallyfar less costly to construct than cell culture facilities. Although thecore oligosaccharide structure transferred to a protein in theendoplasmic reticulum is basically identical in mammals and lowereukaryotes, substantial differences have been found in the subsequentprocessing reactions which occur in the Golgi apparatus of fungi andmammals. In fact, even amongst different lower eukaryotes there exist agreat variety of glycosylation structures. This has historicallyprevented the use of lower eukaryotes as hosts for the production ofrecombinant human glycoproteins despite otherwise notable advantagesover mammalian expression systems.

[0019] Therapeutic glycoproteins produced in a microorganism host suchas yeast utilizing the endogenous host glycosylation pathway differstructurally from those produced in mammalian cells and typically showgreatly reduced therapeutic efficacy. Such glycoproteins are typicallyimmunogenic in humans and show a reduced half-life (and thusbioactivity) in vivo after administration (Takeuchi, 1997). Specificreceptors in humans and animals (i.e., macrophage mannose receptors) canrecognize terminal mannose residues and promote the rapid clearance ofthe foreign glycoprotein from the bloodstream. Additional adverseeffects may include changes in protein folding, solubility,susceptibility to proteases, trafficking, transport,compartmentalization, secretion, recognition by other proteins orfactors, antigenicity, or allergenicity.

[0020] Yeast and filamentous fungi have both been successfully used forthe production of recombinant proteins, both intracellular and secreted(Cereghino, J. L. and J. M. Cregg 2000 FEMS Microbiology Reviews 24(1):45-66; Harkki, A., et al. 1989 Bio-Technology 7(6): 596; Berka, R. M.,et al. 1992 Abstr. Papers Amer. Chem. Soc.203: 121-BIOT; Svetina, M., etal. 2000 J. Biotechnol. 76(2-3): 245-251). Various yeasts, such as K.lactis, Pichia pastoris, Pichia methanolica, and Hansenula polymorpha,have played particularly important roles as eukaryotic expressionsystems because they are able to grow to high cell densities and secretelarge quantities of recombinant protein. Likewise, filamentous fungi,such as Aspergillus niger, Fusarium sp, Neurospora crassa and others,have been used to efficiently produce glycoproteins at the industrialscale. However, as noted above, glycoproteins expressed in any of theseeukaryotic microorganisms differ substantially in N-glycan structurefrom those in animals. This has prevented the use of yeast orfilamentous fungi as hosts for the production of many therapeuticglycoproteins.

[0021] Although glycosylation in yeast and fungi is very different thanin humans, some common elements are shared. The first step, the transferof the core oligosaccharide structure to the nascent protein, is highlyconserved in all eukaryotes including yeast, fungi, plants and humans(compare FIGS. 1A and 1B). Subsequent processing of the coreoligosaccharide, however, differs significantly in yeast and involvesthe addition of several mannose sugars. This step is catalyzed bymannosyltransferases residing in the Golgi (e.g. OCH1, MNT1, MNN1,etc.), which sequentially add mannose sugars to the coreoligosaccharide. The resulting structure is undesirable for theproduction of humanoid proteins and it is thus desirable to reduce oreliminate mannosyltransferase activity. Mutants of S. cerevisiaedeficient in mannosyltransferase activity (e.g. och1 or mnn9 mutants)have shown to be non-lethal and display a reduced mannose content in theoligosaccharide of yeast glycoproteins. Other oligosaccharide processingenzymes, such as mannosylphosphate transferase, may also have to beeliminated depending on the host's particular endogenous glycosylationpattern. After reducing undesired endogenous glycosylation reactions,the formation of complex N-glycans has to be engineered into the hostsystem. This requires the stable expression of several enzymes andsugar-nucleotide transporters. Moreover, one has to localize theseenzymes so that a sequential processing of the maturing glycosylationstructure is ensured.

[0022] Several efforts have been made to modify the glycosylationpathways of eukaryotic microorganisms to provide glycoproteins moresuitable for use as mammalian therapeutic agents. For example, severalglycosyltransferases have been separately cloned and expressed in Scerevisiae (GalT, GnTI), Aspergillus nidulans (GnTI) and other fungi(Yoshida et al., 1999, Kalsner et al., 1995 Glycoconj. J. 12(3):360-370,Schwientek et al., 1995). However, N-glycans resembling those made inhuman cells were not obtained.

[0023] Yeasts produce a variety of mannosyltransferases (e.g.,1,3-mannosyltransferases such as MNN1 in S. cerevisiae; Graham and Emr,1991 J. Cell. Biol. 114(2):207-218), 1,2-mannosyltransferases (e.g.KTR/KRE family from S. cerevisiae), 1,6-mannosyltransferases (e.g., OCHIfrom S. cerevisiae), mannosylphosphate transferases and their regulators(e.g., MNN4 and MNN6 from S. cerevisiae) and additional enzymes that areinvolved in endogenous glycosylation reactions. Many of these genes havebeen deleted individually giving rise to viable organisms having alteredglycosylation profiles. Examples are shown in Table 1. TABLE 1 Examplesof east strains having altered mannosylation N-glycan N-glycan Strain(wild type) Mutation (mutant) Reference S. pombe Man_(>9)GlcNAc₂ OCH1Man₈GlcNAc₂ Yoko-o et al., 2001 FEBS Lett. 489(1): 75-80 S. cerevisiaeMan_(>9)GlcNAc₂ OCH1/ Man₈GlcNAc₂ Nakanishi- MNN1 Shindo et al,. 1993 J.Biol. Chem. 268(35): 26338- 26345 S. cerevisiae Man_(>9)GlcNAc₂ OCH1/Man₈GlcNAc₂ Chiba et al., MNN1/ 1998 J. Biol. MNN4 Chem. 273, 26298-26304 P. pastoris Hyperglyco- OCH1 Not hyper- Welfide, sylated (completeglycosylated Japanese deletion) Application Publication No. 8- 336387 P.pastoris Man_(>8)GlcNAc₂ OCH1 Man_(>8)GlcNAc₂ Contreras (dis- et al. WOruption) 02/00856 A2

[0024] Japanese Patent Application Publication No. 8-336387 disclosesthe deletion of an OCHI homolog in Pichia pastoris. In S. cerevisiae,OCHI encodes a 1,6-mannosyltransferase, which adds a mannose to theglycan structure Man₈GlcNAc₂ to yield Man₉GlcNAc₂. The Man₉GlcNAc₂structure, which contains three 1,6 mannose residues, is then asubstrate for further 1,2-, 1,6-, and 1,3- mannosyltransferases in vivo,leading to the hypermannosylated glycoproteins that are characteristicfor S. cerevisiae and which typically may have 30-40 mannose residuesper N-glycan. Because the Och1p initiates the transfer of 1,6 mannose tothe Man₈GlcNAc₂ core, it is often referred to as the “initiating 1,6mannosyltransferase” to distinguish it from other 1,6mannosyltransferases acting later in the Golgi. In an och1 mnn1 mnn4mutant strain of S. cerevisiae, proteins glycosylated with Man₈GlcNAc₂accumulate and hypermannosylation does not occur. However, Man₈GlcNAc₂is not a substrate for mammalian glycosyltransferases, such as humanUDP-GlcNAc transferase I, and accordingly, the use of that mutantstrain, in itself, is not useful for producing mammalian-like proteins,i.e., with complex or hybrid glycosylation patterns.

[0025] Although Japanese Patent Application Publication No. 8-336387discloses methods to obtain an och1 mutant of P. pastoris displaying areduced mannosylation phenotype, it provides no data on whether theinitiating 1,6 mannosyltransferase activity presumed to be encoded byOCHI is reduced or eliminated. It is well-established in the field offungal genetics that homologs of genes often do not play the same rolein their respective host organism. For example, the Neurospora rca-1gene complements an Aspergillus flbD sporulation mutant but has noidentifiable role in Neurospora sporulation. Shen, W. C. et al.,Genetics 1998;148(3):1031-41. More recently, Contreras (WO 02/00856 A2)shows that, in an och1 mutant of P. pastoris, at least 50% of the cellwall glycans cannot be trimmed to Man₅GlcNAc₂ with a Trichoderma reeseiα-1,2-mannosidase (see FIG. 11 of WO 02/00856 A2). As the wild-typedisplays a very similar glycosylation pattern (FIG. 10, Panel 2 of WO02/00856 A2), it appears that the OCHI gene of P. pastoris may notencode the initiating 1,6-mannosyltransferase activity and is thusdifferent from its genetic homolog in S. cerevisiae. Thus, to date,there is no evidence that initiating α-1,6-mannosyltransferase activityis eliminated in och1 mutants of P. pastoris, which further supports thenotion that the glycosylation pathways of S. cerevisiae and P. pastorisare significantly different.

[0026] Martinet et al. (Biotechnol. Lett. 1998, 20(12), 1171-1177)reported the expression of α-1,2-mannosidase from T. reesei in P.pastoris. Some mannose trimming from the N-glycans of a model proteinwas observed. However, the model protein had no N-glycans with thestructure Man₅GlcNAc₂, which would be necessary as an intermediate forthe generation of complex N-glycans. Accordingly, that system is notuseful for producing proteins with complex or hybrid glycosylationpatterns.

[0027] Similarly, Chiba et al. (1998) expressed α-1,2-mannosidase fromAspergillus saitoi in the yeast Saccharomyces cerevisiae. A signalpeptide sequence (His-Asp-Glu-Leu) was engineered into the exogenousmannosidase to promote its retention in the endoplasmic reticulum. Inaddition, the yeast host was a mutant lacking enzyme activitiesassociated with hypermannosylation of proteins: 1,6-mannosyltransferase(och1); 1,3-mannosyltransferase (mnn1); and a regulator ofmannosylphosphate transferase (mnn4). The N-glycans of the triple mutanthost consisted primarily of the structure Man₈GlcNAc₂, rather than thehigh mannose forms found in wild-type S. cerevisiae. In the presence ofthe engineered mannosidase, the N-glycans of a model protein(carboxypeptidase Y) were trimmed to give a mixture consisting of 27mole % Man₅GlcNAc₂, 22 mole % Man₆GlcNAc₂, 22 mole % Man₇GlcNAc₂, and 29mole % Man₈GlcNAc₂. Trimming of cell wall glycoproteins was lessefficient, with only 10 mole % of the N-glycans having the desiredMan₅GlcNAc₂ structure.

[0028] Even if all the Man₅GlcNAc₂ glycans were the correct Man₅GlcNAc₂form that can be converted to GlcNAcMan₅GlcNAc₂ by GnTI, the abovesystem would not be efficient for the production of proteins havinghuman-like glycosylation patterns. If several glycosylation sites arepresent in a desired protein, the probability (P) of obtaining such aprotein in a correct form follows the relationship P=(F)^(n), where nequals the number of glycosylation sites, and F equals the fraction ofdesired glycoforms. A glycoprotein with three glycosylation sites wouldhave a 0.1% chance of providing the appropriate precursors for complexand hybrid N-glycan processing on all of its glycosylation sites. Thus,using the system of Chiba to make a glycoprotein having a singleN-glycosylation site, at least 73 mole % would have an incorrectstructure. For a glycoprotein having two or three N-glycosylation sites,at least 93 or 98 mole % would have an incorrect structure,respectively. Such low efficiencies of conversion are unsatisfactory forthe production of therapeutic agents, particularly as the separation ofproteins having different glycoforms is typically costly and difficult.

[0029] Chiba et al. (WO 01/14522) have shown high levels of Man₅GlcNAc₂structures on recombinant fibroblast growth factor (FGF), a secretedsoluble glycoprotein produced in S. cerevisiae. It is not clear,however, that the detected Man₅GlcNAc₂ was produced inside the host cell(i.e. in vivo) because the α-1,2 mannosidase was targeted by fusion withan HDEL localization tag, a mechanism, which is known to be leaky(Pelham H. R. (1998) EMBO J. 7,913-918). It is more likely that FGF wassecreted into the medium, where it was then processed by α-1,2mannosidase which had escaped the HDEL retrieval mechanism and leakedinto the medium. As mentioned above, an intracellular protein (CPY),expressed in the same strain, contained mostly glycans (more than 73%)that were Man₆GlcNAc₂ and larger. The majority of the Man₅GlcNAc₂structures on FGF are, thus, likely to have been produced ex vivo. It isfurther unclear whether the Man₅GlcNAc₂ structures that were producedwere productive substrates for GnTI.

[0030] As the above work demonstrates, one can trim Man₈GlcNAc₂structures to a Man₅GlcNAc₂ isomer in S. cerevisiae, although highefficiency trimming greater than 50% in vivo has yet to be determined,by engineering a fungal mannosidase from A. saitoi into the endoplasmicreticulum (ER). The shortcomings of this approach are two-fold: (1) itis not clear whether the Man₅GlcNAc₂ structures formed are in factformed in vivo (rather than having been secreted and further modified bymannosidases outside the cell); and (2) it is not clear whether anyMan₅GlcNAc₂ structures formed, if in fact formed in vivo, are thecorrect isoform to be a productive substrate for subsequent N-glycanmodification by GlcNAc transferase I (Maras et al., 1997, Eur. JBiochem. 249, 701-707).

[0031] With the objective of providing a more human-like glycoproteinderived from a fungal host, U.S. Pat. No. 5,834,251 discloses a methodfor producing a hybrid glycoprotein derived from Trichoderma reseei. Ahybrid N-glycan has only mannose residues on the Manα1-6 arm of the coremannose structure and one or two complex antennae on the Manα1-3 arm.While this structure has utility, the method has the disadvantage thatnumerous enzymatic steps must be performed in vitro, which is costly andtime-consuming. Isolated enzymes are expensive to prepare and needcostly substrates (e.g. UDP-GlcNAc). The method also does not allow forthe production of complex glycans on a desired protein.

Intracellular Mannosidase Activity Involved in N-glycan Trimming

[0032] Alpha-1,2-mannosidase activity is required for the trimming ofMan₈GlcNAc₂ to form Man₅GlcNAc₂, which is a major intermediate forcomplex N-glycan formation in mammals. Previous work has shown thattruncated murine, fungal and human α-1,2-mannosidase can be expressed inthe methylotropic yeast P. pastoris and display Man₈GlcNAc₂ toMan₅GlcNAc₂ trimming activity (Lal et al., Glycobiology 1998October;8(10):981-95; Tremblay et al., Glycobiology 1998June;8(6):585-95, Callewaert et al., 2001). However, to date, no reportsexist that show the high level in vivo trimming of Man₈GlcNAc₂ toMan₅GlcNAc₂ on a secreted glycoprotein from P. pastoris.

[0033] While it is useful to engineer strains that are able to produceMan₅GlcNAc₂ as the primary N-glycan structure, any attempt to furthermodify these high mannose precursor structures to more closely resemblehuman glycans requires additional in vivo or in vitro steps. Methods tofurther humanize glycans from fungal and yeast sources in vitro aredescribed in U.S. Pat. No. 5,834,251 (supra). As discussed above,however, if Man₅GlcNAc₂ is to be further humanized in vivo, one has toensure that the generated Man₅GlcNAc₂ structures are, in fact, generatedintracellularly and not the product of mannosidase activity in themedium. Complex N-glycan formation in yeast or fungi will require highlevels of Man₅GlcNAc₂ to be generated within the cell because onlyintracellular Man₅GlcNAc₂ glycans can be further processed to hybrid andcomplex N-glycans in vivo. In addition, one has to demonstrate that themajority of Man₅GlcNAc₂ structures generated are in fact a substrate forGnTI and thus allow the formation of hybrid and complex N-glycans.

[0034] Moreover, the mere presence of an α-1,2-mannosidase in the celldoes not, by itself, ensure proper intracellular trimming of Man₈GlcNAC₂to Man₅GlcNAc₂. (See, e.g., Contreras et al. WO 02/00856 A2, in which anHDEL tagged mannosidase of T. reesei is localized primarily in the ERand co-expressed with an influenza haemagglutinin (HA) reporter proteinon which virtually no Man₅GlcNAc₂ could be detected. See also Chiba etal., 1998 (supra), in which a chimeric α-1,2-mannosidase/Och1ptransmembrane domain fusion localized in the ER, early Golgi and cytosolof S. cerevisiae, had no mannosidase trimming activity). Accordingly,mere localization of a mannosidase in the ER or Golgi is insufficient toensure activity of the respective enzyme in that targeted organelle.(See also, Martinet et al. (1998), supra, showing that α-1,2-mannosidasefrom T. reesei, while localizing intracellularly, increased rather thandecreased the extent of mannosylation). To date, there is no report thatdemonstrates the intracellular localization of an active heterologousα-1,2-mannosidase in either yeast or fungi using a transmembranelocalization sequence.

[0035] Accordingly, the need exists for methods to produce glycoproteinscharacterized by a high intracellular Man₅GlcNAc₂ content which can befurther processed into human-like glycoprotein structures in non-humaneukaryotic host cells, and particularly in yeast and filamentous fungi.

SUMMARY OF THE INVENTION

[0036] Host cells and cell lines having genetically modifiedglycosylation pathways that allow them to carry out a sequence ofenzymatic reactions which mimic the processing of glycoproteins inmammals, especially in humans, have been developed. Recombinant proteinsexpressed in these engineered hosts yield glycoproteins more similar, ifnot substantially identical, to their mammalian, e.g., humancounterparts. Host cells of the invention, e.g., lower eukaryoticmicro-organisms and other non-human, eukaryotic host cells grown inculture, are modified to produce N-glycans such as Man₅GlcNAc₂ or otherstructures produced along human glycosylation pathways. This is achievedusing a combination of engineering and/or selection of strains which: donot express certain enzymes which create the undesirable structurescharacteristic of the fungal glycoproteins; which express heterologousenzymes selected either to have optimal activity under the conditionspresent in the host cell where activity is to be achieved; orcombinations thereof; wherein the genetically engineered eukaryoteexpresses at least one heterologous enzyme activity required to producea “human-like” glycoprotein. Host cells of the invention may be modifiedfurther by heterologous expression of one or more activities such asglycosyltransferases, sugar transporters and mannosidases, to becomestrains for the production of mammalian, e.g., human therapeuticglycoproteins.

[0037] The present invention thus provides a glycoprotein productionmethod using (1) a lower eukaryotic host such as a unicellular orfilamentous fungus, or (2) any non-human eukaryotic organism that has adifferent glycosylation pattern from humans, to modify the glycosylationcomposition and structures of the proteins made in a host organism(“host cell”) so that they resemble more closely carbohydrate structuresfound in mammalian, e.g., human proteins. The process allows one toobtain an engineered host cell which can be used to express and targetany desirable gene(s), e.g.,one involved in glycosylation, by methodsthat are well-established in the scientific literature and generallyknown to the artisan in the field of protein expression. Host cells withmodified oligosaccharides are created or selected. N-glycans made in theengineered host cells have a Man₅GlcNAc₂ core structure which may thenbe modified further by heterologous expression of one or more enzymes,e.g., glycosyltransferases, glycosidases, sugar transporters andmannosidases, to yield human-like glycoproteins. For the production oftherapeutic proteins, this method may be adapted to engineer cell linesin which any desired glycosylation structure may be obtained.

[0038] Accordingly, in one embodiment, the invention provides a methodfor producing a human-like glycoprotein in a non-human eukaryotic hostcell. The host cell of the invention is selected or engineered to bedepleted in 1,6-mannosyl transferase activities which would otherwiseadd mannose residues onto the N-glycan on a glycoprotein. One or moreenzymes (enzymatic activities) are introduced into the host cell whichenable the production of a Man₅GlcNAc₂ carbohydrate structure at a highyield, e.g., at least 30 mole percent. In a more preferred embodiment,at least 10% of the Man₅GlcNAc₂ produced within the host cell is aproductive substrate for GnTI and thus for further glycosylationreactions in vivo and/or in vitro that produce a finished N-glycan thatis similar or identical to that formed in mammals, especially humans.

[0039] In another embodiment, a nucleic acid molecule encoding one ormore enzymes for production of a Man₅GlcNAc₂ carbohydrate structure isintroduced into a host cell selected or engineered to be depleted in1,6-mannosyltransferase activities. In one preferred embodiment, atleast one enzyme introduced into the host cell is selected to haveoptimal activity at the pH of the subcellular location where thecarbohydrate structure is produced. In another preferred embodiment, atleast one enzyme is targeted to a host subcellular organelle where theenzyme will have optimal activity, e.g., by means of a chimeric proteincomprising a cellular targeting signal peptide not normally associatedwith the enzyme.

[0040] The invention further provides isolated nucleic acid moleculesand vectors comprising such molecules which encode an initiatingα1,6-mannosyl transferase activity isolated from P. pastoris or from K.lactis. These nucleic acid molecules comprise sequences that arehomologous to the OCHI gene in S. cerevisiae. These and homologoussequences are useful for constructing host cells which will nothypermannosylate the N-glycan of a glycoprotein.

[0041] In another embodiment, the host cell is engineered to express aheterologous glycosidase, e.g., by introducing into the host one or morenucleic acid molecules encoding the glycosidase. Preferably, a nucleicacid molecule encodes one or more mannosidase activities involved in theproduction of Man₅GlcNAc₂ from Man₈GlcNAc₂ or Man₉GlcNAc₂. In apreferred embodiment, at least one of the encoded mannosidase activitieshas a pH optimum within 1.4 pH units of the average pH optimum of otherrepresentative enzymes in the organelle in which the mannosidaseactivity is localized, or has optimal activity at a pH of between about5.1 and about 8.0, preferably between about 5.5 and about 7.5.Preferably, the heterologous enzyme is targeted to the endoplasmicreticulum, the Golgi apparatus or the transport vesicles between ER andGolgi of the host organism, where it trims N-glycans such as Man₉GlcNAc₂to yield high levels of Man₅GlcNAc₂. In one embodiment, the enzyme istargeted by forming a fusion protein between a catalytic domain of theenzyme and a cellular targeting signal peptide, e.g., by the in-frameligation of a DNA fragment encoding a cellular targeting signal peptidewith a DNA fragment encoding a glycosylation enzyme or catalyticallyactive fragment thereof.

[0042] In yet another embodiment, the glycosylation pathway of a host ismodified to express a sugar nucleotide transporter. In a preferredembodiment, a nucleotide diphosphatase enzyme is also expressed. Thetransporter and diphosphatase improve the efficiency of engineeredglycosylation steps, by providing the appropriate substrates for theglycosylation enzymes in the appropriate compartments, reducingcompetitive product inhibition, and promoting the removal of nucleosidediphosphates.

[0043] The present invention also provides a combinatorial nucleic acidlibrary useful for making fusion constructs which can target a desiredprotein or polypeptide fragment, e.g., an enzyme involved inglycosylation or a catalytic domain thereof, to a selected subcellularregion of a host cell. In one preferred embodiment, the combinatorialnucleic acid library comprises (a) nucleic acid sequences encodingdifferent cellular targeting signal peptides and (b) nucleic acidsequences encoding different polypeptides to be targeted. Nucleic acidsequences of or derived from (a) and (b) are ligated together to producefusion constructs, at least one of which encodes a functional proteindomain (e.g., a catalytic domain of an enzyme) ligated in-frame to aheterologous cellular targeting signal peptide, i.e., one which itnormally does not associate with.

[0044] The invention also provides a method for modifying theglycosylation pathway of a host cell (e.g., any eukaryotic host cell,including a human host cell) using enzymes involved in modifyingN-glycans including glycosidases and glycosyltransferases; bytransforming the host cell with a nucleic acid (e.g., a combinatorial)library of the invention to produce a genetically mixed cell populationexpressing at least one and preferably two or more distinct chimericglycosylation enzymes having a catalytic domain ligated in-frame to acellular targeting signal peptide which it normally does not associatewith. A host cell having a desired glycosylation phenotype mayoptionally be selected from the population. Host cells modified usingthe library and associated methods of the invention are useful, e.g.,for producing glycoproteins having a glycosylation pattern similar oridentical to those produced in mammals, especially humans.

[0045] In another aspect, the combinatorial library of the presentinvention enables production of one or a combination of catalyticallyactive glycosylation enzymes, which successfully localize tointracellular compartments in which they function efficiently in theglycosylation/secretory pathway. Preferred enzymes convertMan₅(α-1,2-Man)₃₋₉GlcNAc₂ to Man₅GlcNAc₂ at high efficiency in vivo. Inaddition, the invention provides eukaryotic host strains, and inparticular, yeasts, fungal, insect, plant, plant cells, algae and insectcell hosts, capable of producing glycoprotein intermediates or productswith Man₅GlcNAc₂ and/or GlcNAcMan₅GlcNAc₂ as the predominant N-glycan.

[0046] The present invention also provides recombinant molecules derivedfrom a combinatorial nucleic acid library; vectors, including expressionvectors, comprising such recombinant molecules; proteins encoded by therecombinant molecules and vectors; host cells transformed with therecombinant molecules or vectors; glycoproteins produced from suchtransformed hosts; and methods for producing, in vivo, glycoproteinintermdiates or products with predominantly Man₅GlcNAc₂ orGlcNAcMan₅GlcNAc₂ N-glycans covalently attached to appropriateglycosylation sites using the combinatorial library.

[0047] Further aspects of this invention include methods, compositionsand kits for diagnostic and therapeutic uses in which presence orabsence of Man₅GlcNAc₂ and/or GlcNAcMan₅GlcNAc₂ on a glycoprotein may bedetected.

BRIEF DESCRIPTION OF THE DRAWINGS

[0048]FIG. 1A is a schematic diagram of a typical fungal N-glycosylationpathway.

[0049]FIG. 1B is a schematic diagram of a typical human N-glycosylationpathway.

[0050]FIG. 2 depicts construction of a combinatorial DNA library offusion constructs. FIG. 2A diagrams the insertion of a targeting peptidefragment into pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.). FIG. 2B showsthe generated targeting peptide sub-library having restriction sitesNotI-AscI. FIG. 2C diagrams the insertion of a catalytic domain regioninto pJN347, a modified pUC19 vector. FIG. 2D shows the generatedcatalytic domain sub-library having restriction sites NotI, AscI andPacI. FIG. 2E depicts one particular fusion construct generated from thetargeting peptide sub-library and the catalytic domain sub-library.

[0051]FIG. 3 illustrates the M. musculus α-1,2-mannosidase IA openreading frame. The sequences of the PCR primers used to generateN-terminal truncations are underlined.

[0052]FIG. 4 illustrates engineering of vectors with multipleauxotrophic markers and genetic integration of target proteins in the P.pastoris OCHI locus.

[0053] FIGS. 5A-5E show MALDI-TOF analysis demonstrating production ofkringle 3 domain of human plasminogen (K3) glycoproteins havingMan₅GlcNAc₂ as the predominant N-glycan structure in P. pastoris. FIG.5A depicts the standard Man₅GlcNAc₂ [a] glycan (Glyko, Novato, Calif.)and Man₅GlcNAc₂+Na⁺ [b]. FIG. 5B shows PNGase-released glycans from K3wild type. The N-glycans shown are as follows: Man₉GlcNAc₂ [d];Man₁₀GlcNAc₂ [e]; Man₁₁GlcNAc₂ [f]; Man₁₂GlcNAc₂ [g]. FIG. 5C depictsthe och1 deletion resulting in the production of Man₈GlcNAc₂ [c] as thepredominant N-glycan. FIGS. 5D and 5E show the production of Man₅GlcNAc₂[b] after in vivo trimming of Man₈GlcNAc₂ with a chimericα-1,2-mannosidase. The predominant N-glycan is indicated by a peak witha mass (m/z) of 1253 consistent with its identification as Man₅GlcNAc₂[b].

[0054] FIGS. 6A-6F show MALDI-TOF analysis demonstrating production ofIFN-β glycoproteins having Man₅GlcNAc₂ as the predominant N-glycanstructure in P. pastoris. FIG. 6A shows the standard Man₅GlcNAc₂ [a] andMan₅GlcNAc₂+Na⁺ [b] as the standard (Glyko, Novato, Calif.). FIG. 6Bshows PNGase-released glycans from IFN-β wildtype. FIG. 6C depicts theoch1 knock-out producing Man₈GlcNAc₂ [C]; Man₉GlcNAc₂ [d]; Man₁₀GlcNAc₂[e]; Man₁₁GlcNAC₂ [f]; Man₁₂GlcNAc₂ [g]; and no production ofMan₅GlcNAc₂ [b]. FIG. 6D shows relatively small amount of Man₅GlcNAc₂[b] among other intermediate N-glycans Man₈GlcNAC₂ [c] to Man₁₂GlcNAc₂[g]. FIG. 6E shows a significant amount of Man₅GlcNAc₂ [b] relative tothe other glycans Man₈GlcNAc₂ [c] and Man₉GlcNAc₂ [d] produced by pGC5(Saccharomyces MNS1(m)/mouse mannosidase IB Δ99). FIG. 6F showspredominant production of Man₅GlcNAc₂ [b] on the secreted glycoproteinIFN-β by pFB8 (Saccharomyces SEC12 (m)/mouse mannosidase IA Δ187). TheN-glycan is indicated by a peak with a mass (m/z) of 1254 consistentwith its identification as Man₅GlcNAC₂ [b].

[0055]FIG. 7 shows a high performance liquid chromatogram for: (A)Man₉GlcNAc₂ standard labeled with 2-AB (negative control); (B)supernatant of medium P. pastoris, Δoch1 transformed with pFB8mannosidase, which demonstrates a lack of extracellular mannosidaseactivity in the supernatant; and (C) Man₉GlcNAc₂ standard labeled with2-AB after exposure to T. reesei mannosidase (positive control).

[0056]FIG. 8 shows a high performance liquid chromatogram for: (A)Man₉GlcNAc₂ standard labeled with 2-AB (negative control); (B)supernatant of medium P. pastoris, Δoch1 transformed with pGC5mannosidase, which demonstrates a lack of extracellular mannosidaseactivity in the supernatant; and (C) Man₉GlcNAc₂ standard labeled with2-AB after exposure to T. reesei mannosidase (positive control).

[0057]FIG. 9 shows a high performance liquid chromatogram for: (A)Man₉GlcNAc₂ standard labeled with 2-AB (negative control); (B)supernatant of medium P. pastoris, Δoch1 transformed with pBC18-5mannosidase, which demonstrates lack of extracellular mannosidaseactivity in the supernatant; and (C) supernatant of medium P. pastoris,Δoch1 transformed with pDD28-3, which demonstrates activity in thesupernatant (positive control).

[0058] Figures 10A-10B demonstrate the activity of an UDP-GlcNActransporter in the production of GlcNAcMan₅GlcNAc₂ in P. pastoris. FIG.10A depicts a P. pastoris strain (YSH-3) with a human GnTI but withoutthe UDP-GlcNAc transporter resulting in some production ofGlcNAcMan₅GlcNAc₂ [b] but a predominant production of Man₅GlcNAc₂ [a].FIG. 10B depicts the addition of UDP-GlcNAc transporter from K. lactisin a strain (PBP-3) with the human GnTI, which resulted in thepredominant production of GlcNAcMan₅GlcNAc₂ [b]. The single prominentpeak of mass (m/z) at 1457 is consistent with its identification asGlcNAcMan₅GlcNAc₂ [b] as shown in FIG. 10B.

[0059]FIG. 11 shows a pH optimum of a heterologous mannosidase enzymeencoded by pBB27-2 (Saccharomyces MNN10 (s)/C. elegans mannosidase IBΔ31) expressed in P. pastoris.

[0060] FIGS. 12A-12C show MALDI-TOF analysis of N-glycans released froma cell free extract of K. lactis. FIG. 12A shows the N-glycans releasedfrom wild-type cells, which includes high-mannose type N-glycans. FIG.12B shows the N-glycans released from och1 mnn1 deleted cells, revealinga distinct peak of mass (m/z) at 1908 consistent with its identificationas Man₉GlcNAc₂ [d]. FIG. 12C shows the N-glycans released from och1 mnn1deleted cells after in vitro α-1,2-mannosidase digest corresponding to apeak consistent with Man₅GlcNAc₂.

[0061]FIG. 13 represents T-DNA cassettes with catalytic domain(s) ofglycosylation enzymes fused in-frame to different leader sequences. Theends of the T-DNA are marked by the right (rb) and left borders (lb).Various promoters and terminators may also be used. The plant selectablemarker can also be varied. The right and left borders are required onlyfor agrobacterium-mediated transformation and not for particlebombardment or electroporation.

DETAILED DESCRIPTION OF THE INVENTION

[0062] Unless otherwise defined herein, scientific and technical termsused in connection with the present invention shall have the meaningsthat are commonly understood by those of ordinary skill in the art.Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art. Generally,nomenclatures used in connection with, and techniques of biochemistry,enzymology, molecular and cellular biology, microbiology, genetics andprotein and nucleic acid chemistry and hybridization described hereinare those well-known and commonly used in the art.

[0063] The methods and techniques of the present invention are generallyperformed according to conventional methods well-known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al. Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates (1992, and Supplements to 2002); Harlow andLane Antibodies: A Laboratory Manual Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1990); Introduction to Glycobiology,Maureen E. Taylor, Kurt Drickamer, Oxford Univ. Press (2003);Worthington Enzyme Manual, Worthington Biochemical Corp. Freehold, N.J.;Handbook of Biochemistry: Section A Proteins Vol I 1976 CRC Press;Handbook of Biochemistry: Section A Proteins Vol II 1976 CRC Press;Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999).The nomenclatures used in connection with, and the laboratory proceduresand techniques of, molecular and cellular biology, protein biochemistry,enzymology and medicinal and pharmaceutical chemistry described hereinare those well known and commonly used in the art.

[0064] All publications, patents and other references mentioned hereinare incorporated by reference.

[0065] The following terms, unless otherwise indicated, shall beunderstood to have the following meanings:

[0066] As used herein, the term “N-glycan” refers to an N-linkedoligosaccharide, e.g., one that is attached by anasparagine-N-acetylglucosamine linkage to an asparagine residue of apolypeptide. N-glycans have a common pentasaccharide core of Man₃GlcNAc₂(“Man” refers to mannose; “Glc” refers to glucose; and “NAc” refers toN-acetyl; GlcNAc refers to N-acetylglucosamine). The term “trimannosecore” used with respect to the N-glycan also refers to the structureMan₃GlcNAc₂ (“Man₃”). N-glycans differ with respect to the number ofbranches (antennae) comprising peripheral sugars (e.g., fucose andsialic acid) that are added to the Man₃ core structure. N-glycans areclassified according to their branched constituents (e.g., high mannose,complex or hybrid).

[0067] A “high mannose” type N-glycan has five or more mannose residues.A “complex” type N-glycan typically has at least one GlcNAc attached tothe 1,3 mannose arm and at least one GlcNAc attached to the 1,6 mannosearm of the trimannose core. Complex N-glycans may also have galactose(“Gal”) residues that are optionally modified with sialic acid orderivatives (“NeuAc”, where “Neu” refers to neuraminic acid and “Ac”refers to acetyl). A complex N-glycan typically has at least one branchthat terminates in an oligosaccharide such as, for example: NeuNAc-;NeuAca2-6GalNAca1-; NeuAca2-3Galb1-3GalNAca1-;NeuAca2-3/6Galb1-4GlcNAcb1-; GlcNAca1-4Galb1-(mucins only);Fuca1-2Galb1-(blood group H). Sulfate esters can occur on galactose,GalNAc, and GlcNAc residues, and phosphate esters can occur on mannoseresidues. NeuAc (Neu: neuraminic acid; Ac:acetyl) can be O-acetylated orreplaced by NeuGI (N-glycolylneuraminic acid). Complex N-glycans mayalso have intrachain substitutions comprising “bisecting” GlcNAc andcore fucose (“Fuc”). A “hybrid” N-glycan has at least one GlcNAc on theterminal of the 1,3 mannose arm of the trimannose core and zero or moremannoses on the 1,6 mannose arm of the trimannose core.

[0068] The term “predominant” or “predominantly” used with respect tothe production of N-glycans refers to a structure which represents themajor peak detected by matrix assisted laser desorption ionization timeof flight mass spectrometry (MALDI-TOF) analysis.

[0069] Abbreviations used herein are of common usage in the art, see,e.g., abbreviations of sugars, above. Other common abbreviations include“PNGase”, which refers to peptide N-glycosidase F (EC 3.2.2.18); “GlcNAcTr” or “GnT,” which refers to N-acetylglucosaminyl Transferase enzymes;“NANA” refers to N-acetylneuraminic acid.

[0070] As used herein, a “humanized glycoprotein” or a “human-likeglycoprotein” refers alternatively to a protein having attached theretoN-glycans having less than four mannose residues, and syntheticglycoprotein intermediates (which are also useful and can be manipulatedfurther in vitro or in vivo) having at least five mannose residues.Preferably, glycoproteins produced according to the invention contain atleast 30 mole %, preferably at least 40 mole % and more preferably50-100 mole % of the Man₅GlcNAc₂ intermediate, at least transiently.This may be achieved, e.g., by engineering a host cell of the inventionto express a “better”, i.e., a more efficient glycosylation enzyme. Forexample, a mannosidase is selected such that it will have optimalactivity under the conditions present at the site in the host cell whereproteins are glycosylated and is introduced into the host cellpreferably by targeting the enzyme to a host cell organelle whereactivity is desired.

[0071] The term “enzyme”, when used herein in connection with alteringhost cell glycosylation, refers to a molecule having at least oneenzymatic activity, and includes full-length enzymes, catalyticallyactive fragments, chimerics, complexes, and the like. A “catalyticallyactive fragment” of an enzyme refers to a polypeptide having adetectable level of functional (enzymatic) activity.

[0072] A lower eukaryotic host cell, when used herein in connection withglycosylation profiles, refers to any eukaryotic cell which ordinarilyproduces high mannose containing N-glycans, and thus is meant to includesome animal or plant cells and most typical lower eukaryotic cells,including uni- and multicellular fungal and algal cells.

[0073] As used herein, the term “secretion pathway” refers to theassembly line of various glycosylation enzymes to which a lipid-linkedoligosaccharide precursor and an N-glycan substrate are sequentiallyexposed, following the molecular flow of a nascent polypeptide chainfrom the cytoplasm to the endoplasmic reticulum (ER) and thecompartments of the Golgi apparatus. Enzymes are said to be localizedalong this pathway. An enzyme X that acts on a lipid-linked glycan or anN-glycan before enzyme Y is said to be or to act “upstream” to enzyme Y;similarly, enzyme Y is or acts “downstream” from enzyme X.

[0074] The term “targeting peptide” as used herein refers to nucleotideor amino acid sequences encoding a cellular targeting signal peptidewhich mediates the localization (or retention) of an associated sequenceto sub-cellular locations, e.g., organelles.

[0075] The term “polynucleotide” or “nucleic acid molecule” refers to apolymeric form of nucleotides of at least 10 bases in length. The termincludes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNAmolecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA orRNA containing non-natural nucleotide analogs, non-native intemucleosidebonds, or both. The nucleic acid can be in any topological conformation.For instance, the nucleic acid can be single-stranded, double-stranded,triple-stranded, quadruplexed, partially double-stranded, branched,hairpinned, circular, or in a padlocked conformation. The term includessingle and double stranded forms of DNA. A nucleic acid molecule of thisinvention may include both sense and antisense strands of RNA, cDNA,genomic DNA, and synthetic forms and mixed polymers of the above. Theymay be modified chemically or biochemically or may contain non-naturalor derivatized nucleotide bases, as will be readily appreciated by thoseof skill in the art. Such modifications include, for example, labels,methylation, substitution of one or more of the naturally occurringnucleotides with an analog, intemucleotide modifications such asuncharged linkages (e.g., methyl phosphonates, phosphotriesters,phosphoramidates, carbamates, etc.), charged linkages (e.g.,phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g.,polypeptides), intercalators (e.g., acridine, psoralen, etc.),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids, etc.) Also included are synthetic molecules that mimicpolynucleotides in their ability to bind to a designated sequence viahydrogen bonding and other chemical interactions. Such molecules areknown in the art and include, for example, those in which peptidelinkages substitute for phosphate linkages in the backbone of themolecule.

[0076] Unless otherwise indicated, a “nucleic acid comprising SEQ IDNO:X” refers to a nucleic acid, at least a portion of which has either(i) the sequence of SEQ ID NO:X, or (ii) a sequence complementary to SEQID NO:X. The choice between the two is dictated by the context. Forinstance, if the nucleic acid is used as a probe, the choice between thetwo is dictated by the requirement that the probe be complementary tothe desired target.

[0077] An “isolated” or “substantially pure” nucleic acid orpolynucleotide (e.g., an RNA, DNA or a mixed polymer) is one which issubstantially separated from other cellular components that naturallyaccompany the native polynucleotide in its natural host cell, e.g.,ribosomes, polymerases, and genomic sequences with which it is naturallyassociated. The term embraces a nucleic acid or polynucleotide that (1)has been removed from its naturally occurring environment, (2) is notassociated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (3) is operatively linkedto a polynucleotide which it is not linked to in nature, or (4) does notoccur in nature. The term “isolated” or “substantially pure” also can beused in reference to recombinant or cloned DNA isolates, chemicallysynthesized polynucleotide analogs, or polynucleotide analogs that arebiologically synthesized by heterologous systems.

[0078] However, “isolated” does not necessarily require that the nucleicacid or polynucleotide so described has itself been physically removedfrom its native environment. For instance, an endogenous nucleic acidsequence in the genome of an organism is deemed “isolated” herein if aheterologous sequence (i.e., a sequence that is not naturally adjacentto this endogenous nucleic acid sequence) is placed adjacent to theendogenous nucleic acid sequence, such that the expression of thisendogenous nucleic acid sequence is altered. By way of example, anon-native promoter sequence can be substituted (e.g., by homologousrecombination) for the native promoter of a gene in the genome of ahuman cell, such that this gene has an altered expression pattern. Thisgene would now become “isolated” because it is separated from at leastsome of the sequences that naturally flank it.

[0079] A nucleic acid is also considered “isolated” if it contains anymodifications that do not naturally occur to the corresponding nucleicacid in a genome. For instance, an endogenous coding sequence isconsidered “isolated” if it contains an insertion, deletion or a pointmutation introduced artificially, e.g., by human intervention. An“isolated nucleic acid” also includes a nucleic acid integrated into ahost cell chromosome at a heterologous site, a nucleic acid constructpresent as an episome. Moreover, an “isolated nucleic acid” can besubstantially free of other cellular material, or substantially free ofculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized.

[0080] As used herein, the phrase “degenerate variant” of a referencenucleic acid sequence encompasses nucleic acid sequences that can betranslated, according to the standard genetic code, to provide an aminoacid sequence identical to that translated from the reference nucleicacid sequence.

[0081] The term “percent sequence identity” or “identical” in thecontext of nucleic acid sequences refers to the residues in the twosequences which are the same when aligned for maximum correspondence.The length of sequence identity comparison may be over a stretch of atleast about nine nucleotides, usually at least about 20 nucleotides,more usually at least about 24 nucleotides, typically at least about 28nucleotides, more typically at least about 32 nucleotides, andpreferably at least about 36 or more nucleotides. There are a number ofdifferent algorithms known in the art which can be used to measurenucleotide sequence identity. For instance, polynucleotide sequences canbe compared using FASTA, Gap or Bestfit, which are programs in WisconsinPackage Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTAprovides alignments and percent sequence identity of the regions of thebest overlap between the query and search sequences (Pearson, 1990,herein incorporated by reference). For instance, percent sequenceidentity between nucleic acid sequences can be determined using FASTAwith its default parameters (a word size of 6 and the NOPAM factor forthe scoring matrix) or using Gap with its default parameters as providedin GCG Version 6.1, herein incorporated by reference.

[0082] The term “substantial homology” or “substantial similarity,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 50%, more preferably 60%of the nucleotide bases, usually at least about 70%, more usually atleast about 80%, preferably at least about 90%, and more preferably atleast about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, asmeasured by any well-known algorithm of sequence identity, such asFASTA, BLAST or Gap, as discussed above.

[0083] Alternatively, substantial homology or similarity exists when anucleic acid or fragment thereof hybridizes to another nucleic acid, toa strand of another nucleic acid, or to the complementary strandthereof, under stringent hybridization conditions. “Stringenthybridization conditions” and “stringent wash conditions” in the contextof nucleic acid hybridization experiments depend upon a number ofdifferent physical parameters. Nucleic acid hybridization will beaffected by such conditions as salt concentration, temperature,solvents, the base composition of the hybridizing species, length of thecomplementary regions, and the number of nucleotide base mismatchesbetween the hybridizing nucleic acids, as will be readily appreciated bythose skilled in the art. One having ordinary skill in the art knows howto vary these parameters to achieve a particular stringency ofhybridization.

[0084] In general, “stringent hybridization” is performed at about 25°C. below the thermal melting point (T_(m)) for the specific DNA hybridunder a particular set of conditions. “Stringent washing” is performedat temperatures about 5° C. lower than the T_(m) for the specific DNAhybrid under a particular set of conditions. The T_(m) is thetemperature at which 50% of the target sequence hybridizes to aperfectly matched probe. See Sambrook et al., supra, page 9.51, herebyincorporated by reference. For purposes herein, “high stringencyconditions” are defined for solution phase hybridization as aqueoushybridization (i.e., free of formamide) in 6×SSC (where 20×SSC contains3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65° C. for 8-12 hours,followed by two washes in 0.2×SSC, 0.1% SDS at 65° C. for 20 minutes. Itwill be appreciated by the skilled artisan that hybridization at 65° C.will occur at different rates depending on a number of factors includingthe length and percent identity of the sequences which are hybridizing.

[0085] The term “mutated” when applied to nucleic acid sequences meansthat nucleotides in a nucleic acid sequence may be inserted, deleted orchanged compared to a reference nucleic acid sequence. A singlealteration may be made at a locus (a point mutation) or multiplenucleotides may be inserted, deleted or changed at a single locus. Inaddition, one or more alterations may be made at any number of lociwithin a nucleic acid sequence. A nucleic acid sequence may be mutatedby any method known in the art including but not limited to mutagenesistechniques such as “error-prone PCR” (a process for performing PCR underconditions where the copying fidelity of the DNA polymerase is low, suchthat a high rate of point mutations is obtained along the entire lengthof the PCR product. See, e.g., Leung, D. W., et al., Technique, 1, pp.11-15 (1989) and Caldwell, R. C. & Joyce G. F., PCR Methods Applic., 2,pp. 28-33 (1992)); and “oligonucleotide-directed mutagenesis” (a processwhich enables the generation of site-specific mutations in any clonedDNA segment of interest. See, e.g., Reidhaar-Olson, J. F. & Sauer, R.T., et al., Science, 241, pp. 53-57 (1988)).

[0086] The term “vector” as used herein is intended to refer to anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. One type of vector is a “plasmid”, whichrefers to a circular double stranded DNA loop into which additional DNAsegments may be ligated. Other vectors include cosmids, bacterialartificial chromosomes (BAC) and yeast artificial chromosomes (YAC).Another type of vector is a viral vector, wherein additional DNAsegments may be ligated into the viral genome (discussed in more detailbelow). Certain vectors are capable of autonomous replication in a hostcell into which they are introduced (e.g., vectors having an origin ofreplication which functions in the host cell). Other vectors can beintegrated into the genome of a host cell upon introduction into thehost cell, and are thereby replicated along with the host genome.Moreover, certain preferred vectors are capable of directing theexpression of genes to which they are operatively linked. Such vectorsare referred to herein as “recombinant expression vectors” (or simply,“expression vectors”).

[0087] “Operatively linked” expression control sequences refers to alinkage in which the expression control sequence is contiguous with thegene of interest to control the gene of interest, as well as expressioncontrol sequences that act in trans or at a distance to control the geneof interest.

[0088] The term “expression control sequence” as used herein refers topolynucleotide sequences which are necessary to affect the expression ofcoding sequences to which they are operatively linked. Expressioncontrol sequences are sequences which control the transcription,post-transcriptional events and translation of nucleic acid sequences.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (e.g., ribosome binding sites); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence. The term “control sequences” is intended toinclude, at a minimum, all components whose presence is essential forexpression, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences.

[0089] The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a nucleic acid such asa recombinant vector has been introduced. It should be understood thatsuch terms are intended to refer not only to the particular subject cellbut to the progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. A recombinant host cell may be an isolated cell or cellline grown in culture or may be a cell which resides in a living tissueor organism.

[0090] The term “peptide” as used herein refers to a short polypeptide,e.g., one that is typically less than about 50 amino acids long and moretypically less than about 30 amino acids long. The term as used hereinencompasses analogs and mimetics that mimic structural and thusbiological function.

[0091] The term “polypeptide” as used herein encompasses bothnaturally-occurring and non-naturally-occurring proteins, and fragments,mutants, derivatives and analogs thereof. A polypeptide may be monomericor polymeric. Further, a polypeptide may comprise a number of differentdomains each of which has one or more distinct activities.

[0092] The term “isolated protein” or “isolated polypeptide” is aprotein or polypeptide that by virtue of its origin or source ofderivation (1) is not associated with naturally associated componentsthat accompany it in its native state, (2) when it exists in a puritynot found in nature, where purity can be adjudged with respect to thepresence of other cellular material (e.g., is free of other proteinsfrom the same species) (3) is expressed by a cell from a differentspecies, or (4) does not occur in nature (e.g., it is a fragment of apolypeptide found in nature or it includes amino acid analogs orderivatives not found in nature or linkages other than standard peptidebonds). Thus, a polypeptide that is chemically synthesized orsynthesized in a cellular system different from the cell from which itnaturally originates will be “isolated” from its naturally associatedcomponents. A polypeptide or protein may also be rendered substantiallyfree of naturally associated components by isolation, using proteinpurification techniques well-known in the art. As thus defined,“isolated” does not necessarily require that the protein, polypeptide,peptide or oligopeptide so described has been physically removed fromits native environment.

[0093] The term “polypeptide fragment” as used herein refers to apolypeptide that has an amino-terminal and/or carboxy-terminal deletioncompared to a full-length polypeptide. In a preferred embodiment, thepolypeptide fragment is a contiguous sequence in which the amino acidsequence of the fragment is identical to the corresponding positions inthe naturally-occurring sequence. Fragments typically are at least 5, 6,7, 8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18amino acids long, more preferably at least 20 amino acids long, morepreferably at least 25, 30, 35, 40 or 45, amino acids, even morepreferably at least 50 or 60 amino acids long, and even more preferablyat least 70 amino acids long.

[0094] A “modified derivative” refers to polypeptides or fragmentsthereof that are substantially homologous in primary structural sequencebut which include, e.g., in vivo or in vitro chemical and biochemicalmodifications or which incorporate amino acids that are not found in thenative polypeptide. Such modifications include, for example,acetylation, carboxylation, phosphorylation, glycosylation,ubiquitination, labeling, e.g., with radionuclides, and variousenzymatic modifications, as will be readily appreciated by those wellskilled in the art. A variety of methods for labeling polypeptides andof substituents or labels useful for such purposes are well-known in theart, and include radioactive isotopes such as ¹²⁵I, ³²P, ³⁵S, and ³H,ligands which bind to labeled antiligands (e.g., antibodies),fluorophores, chemiluminescent agents, enzymes, and antiligands whichcan serve as specific binding pair members for a labeled ligand. Thechoice of label depends on the sensitivity required, ease of conjugationwith the primer, stability requirements, and available instrumentation.Methods for labeling polypeptides are well-known in the art. See Ausubelet al., 1992, hereby incorporated by reference.

[0095] A “polypeptide mutant” or “mutein” refers to a polypeptide whosesequence contains an insertion, duplication, deletion, rearrangement orsubstitution of one or more amino acids compared to the amino acidsequence of a native or wild type protein. A mutein may have one or moreamino acid point substitutions, in which a single amino acid at aposition has been changed to another amino acid, one or more insertionsand/or deletions, in which one or more amino acids are inserted ordeleted, respectively, in the sequence of the naturally-occurringprotein, and/or truncations of the amino acid sequence at either or boththe amino or carboxy termini. A mutein may have the same but preferablyhas a different biological activity compared to the naturally-occurringprotein. For instance, a mutein may have an increased or decreasedneuron or NgR binding activity. In a preferred embodiment of the presentinvention, a MAG derivative that is a mutein (e.g., in MAG Ig-likedomain 5) has decreased neuronal growth inhibitory activity compared toendogenous or soluble wild-type MAG.

[0096] A mutein has at least 70% overall sequence homology to itswild-type counterpart. Even more preferred are muteins having 80%, 85%or 90% overall sequence homology to the wild-type protein. In an evenmore preferred embodiment, a mutein exhibits 95% sequence identity, evenmore preferably 97%, even more preferably 98% and even more preferably99% overall sequence identity. Sequence homology may be measured by anycommon sequence analysis algorithm, such as Gap or Bestfit.

[0097] Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinity or enzymatic activity, and (5) confer or modify otherphysicochemical or functional properties of such analogs.

[0098] As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis(2^(nd) Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), which is incorporated herein by reference.Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α-, α-disubstituted amino acids,N-alkyl amino acids, and other unconventional amino acids may also besuitable components for polypeptides of the present invention. Examplesof unconventional amino acids include: 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N-acetylserine, N-fornylmethionine, 3-methylhistidine,5-hydroxylysine, s-N-methylarginine, and other similar amino acids andimino acids (e.g., 4-hydroxyproline). In the polypeptide notation usedherein, the left-hand direction is the amino terminal direction and theright hand direction is the carboxy-terminal direction, in accordancewith standard usage and convention.

[0099] A protein has “homology” or is “homologous” to a second proteinif the nucleic acid sequence that encodes the protein has a similarsequence to the nucleic acid sequence that encodes the second protein.Alternatively, a protein has homology to a second protein if the twoproteins have “similar” amino acid sequences. (Thus, the term“homologous proteins” is defined to mean that the two proteins havesimilar amino acid sequences). In a preferred embodiment, a homologousprotein is one that exhibits 60% sequence homology to the wild typeprotein, more preferred is 70% sequence homology. Even more preferredare homologous proteins that exhibit 80%, 85% or 90% sequence homologyto the wild type protein. In a yet more preferred embodiment, ahomologous protein exhibits 95%, 97%, 98% or 99% sequence identity. Asused herein, homology between two regions of amino acid sequence(especially with respect to predicted structural similarities) isinterpreted as implying similarity in function.

[0100] When “homologous” is used in reference to proteins or peptides,it is recognized that residue positions that are not identical oftendiffer by conservative amino acid substitutions. A “conservative aminoacid substitution” is one in which an amino acid residue is substitutedby another amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of homology may be adjusted upwardsto correct for the conservative nature of the substitution. Means formaking this adjustment are well known to those of skill in the art (see,e.g., Pearson et al., 1994, herein incorporated by reference).

[0101] The following six groups each contain amino acids that areconservative substitutions for one another: 1) Serine (S), Threonine(T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Alanine (A), Valine (V), and 6) Phenylalanine (F),Tyrosine (Y), Tryptophan (W).

[0102] Sequence homology for polypeptides, which is also referred to aspercent sequence identity, is typically measured using sequence analysissoftware. See, e.g., the Sequence Analysis Software Package of theGenetics Computer Group (GCG), University of Wisconsin BiotechnologyCenter, 910 University Avenue, Madison, Wis. 53705. Protein analysissoftware matches similar sequences using measure of homology assigned tovarious substitutions, deletions and other modifications, includingconservative amino acid substitutions. For instance, GCG containsprograms such as “Gap” and “Bestfit” which can be used with defaultparameters to determine sequence homology or sequence identity betweenclosely related polypeptides, such as homologous polypeptides fromdifferent species of organisms or between a wild type protein and amutein thereof. See, e.g., GCG Version 6.1.

[0103] A preferred algorithm when comparing a inhibitory moleculesequence to a database containing a large number of sequences fromdifferent organisms is the computer program BLAST (Altschul, S. F. etal. (1990) J. Mol. Biol. 215:403-410; Gish and States (1993) NatureGenet. 3:266-272; Madden, T. L. et al. (1996) Meth. Enzymol.266:131-141; Altschul, S. F. et al. (1997) Nucleic Acids Res.25:3389-3402; Zhang, J. and Madden, T. L. (1997) Genome Res. 7:649-656),especially blastp or tblastn (Altschul et al., 1997). Preferredparameters for BLASTp are: Expectation value: 10 (default); Filter: seg(default); Cost to open a gap: 11 (default); Cost to extend a gap: 1(default); Max. alignments: 100 (default); Word size: 11 (default); No.of descriptions: 100 (default); Penalty Matrix: BLOWSUM62.

[0104] The length of polypeptide sequences compared for homology willgenerally be at least about 16 amino acid residues, usually at leastabout 20 residues, more usually at least about 24 residues, typically atleast about 28 residues, and preferably more than about 35 residues.When searching a database containing sequences from a large number ofdifferent organisms, it is preferable to compare amino acid sequences.Database searching using amino acid sequences can be measured byalgorithms other than blastp known in the art. For instance, polypeptidesequences can be compared using FASTA, a program in GCG Version 6.1.FASTA provides alignments and percent sequence identity of the regionsof the best overlap between the query and search sequences (Pearson,1990, herein incorporated by reference). For example, percent sequenceidentity between amino acid sequences can be determined using FASTA withits default parameters (a word size of 2 and the PAM250 scoring matrix),as provided in GCG Version 6.1, herein incorporated by reference.

[0105] The term “fusion protein” refers to a polypeptide comprising apolypeptide or fragment coupled to heterologous amino acid sequences.Fusion proteins are useful because they can be constructed to containtwo or more desired functional elements from two or more differentproteins. A fusion protein comprises at least 10 contiguous amino acidsfrom a polypeptide of interest, more preferably at least 20 or 30 aminoacids, even more preferably at least 40, 50 or 60 amino acids, yet morepreferably at least 75, 100 or 125 amino acids. Fusion proteins can beproduced recombinantly by constructing a nucleic acid sequence whichencodes the polypeptide or a fragment thereof in-frame with a nucleicacid sequence encoding a different protein or peptide and thenexpressing the fusion protein. Alternatively, a fusion protein can beproduced chemically by crosslinking the polypeptide or a fragmentthereof to another protein.

[0106] The term “region” as used herein refers to a physicallycontiguous portion of the primary structure of a biomolecule. In thecase of proteins, a region is defined by a contiguous portion of theamino acid sequence of that protein.

[0107] The term “domain” as used herein refers to a structure of abiomolecule that contributes to a known or suspected function of thebiomolecule. Domains may be co-extensive with regions or portionsthereof; domains may also include distinct, non-contiguous regions of abiomolecule. Examples of protein domains include, but are not limitedto, an Ig domain, an extracellular domain, a transmembrane domain, and acytoplasmic domain.

[0108] As used herein, the term “molecule” means any compound,including, but not limited to, a small molecule, peptide, protein,sugar, nucleotide, nucleic acid, lipid, etc., and such a compound can benatural or synthetic.

[0109] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. Exemplary methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ofthe present invention and will be apparent to those of skill in the art.All publications and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control. The materials,methods, and examples are illustrative only and not intended to belimiting.

[0110] Throughout this specification and claims, the word “comprise” orvariations such as “comprises” or “comprising”, will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

Methods for Producing Host Cells Having Man₅GlcNAc₂ ModifiedOligosaccharides for the Generation of Human-like N-Glycans

[0111] The invention provides a method for producing a glycoproteinhaving human-like glycosylation in a non-human eukaryotic host cell. Asdescribed in more detail below, a eukaryotic host cell that does notnaturally express, or which is engineered not to express, one or moreenzymes involved in production of high mannose structures is selected asa starting host cell. Such a selected host cell is engineered to expressone or more enzymes or other factors required to produce human-likeglycoproteins. A desired host strain can be engineered one enzyme ormore than one enzyme at a time. In addition, a nucleic acid moleculeencoding one or more enzymes or activities may be used to engineer ahost strain of the invention. Preferably, a library of nucleic acidmolecules encoding potentially useful enzymes (e.g., chimeric enzymescomprising a catalytically active enzyme fragment ligated in-frame to aheterologous subcellular targeting sequence) is created (e.g., byligation of sub-libraries comprising enzymatic fragments and subcellulartargeting sequences), and a strain having one or more enzymes withoptimal activities or producing the most “human-like” glycoproteins maybe selected by transforming target host cells with one or more membersof the library.

[0112] In particular, the methods described herein enable one to obtain,in vivo, Man₅GlcNAc₂ structures in high yield, at least transiently, forthe purpose of further modifying it to yield complex N-glycans. Asuccessful scheme to obtain suitable Man₅GlcNAc₂ structures inappropriate yields in a host cell, such as a lower eukaryotic organism,generally involves two parallel approaches: (1) reducing high mannosestructures made by endogenous mannosyltransferase activities, if any,and (2) removing 1,2-α-mannose by mannosidases to yield high levels ofsuitable Man₅GlcNAc₂ structures which may be further reacted inside thehost cell to form complex, human-like glycoforms.

[0113] Accordingly, a first step involves the selection or creation of aeukaryotic host cell, e.g., a lower eukaryote, capable of producing aspecific precursor structure of Man₅GlcNAc₂ that is able to accept invivo GlcNAc by the action of a GlcNAc transferase I (“GnTI”). In oneembodiment, the method involves making or using a non-human eukaryotichost cell depleted in a 1,6 mannosyltransferase activity with respect tothe N-glycan on a glycoprotein. Preferably, the host cell is depleted inan initiating 1,6 mannosyltransferase activity (see below). Such a hostcell will lack one or more enzymes involved in the production of highmannose structures which are undesirable for producing human-likeglycoproteins.

[0114] One or more enzyme activities are then introduced into such ahost cell to produce N-glycans within the host cell characterized byhaving at least 30 mol % of the Man₅GlcNAc₂ (“Man₅”) carbohydratestructures. Man₅GlcNAc₂ structures are necessary for complex N-glycanformation: Man₅GlcNAc₂ must be formed in vivo in a high yield (e.g., inexcess of 30%), at least transiently, as subsequent mammalian- andhuman-like glycosylation reactions require Man₅GlcNAc₂ or a derivativethereof.

[0115] This step also requires the formation of a particular isomericstructure of Man₅GlcNAc₂ within the cell at a high yield. WhileMan₅GlcNAc₂ structures are necessary for complex N-glycan formation,their presence is by no means sufficient. That is because Man₅GlcNAc₂may occur in different isomeric forms, which may or may not serve as asubstrate for GlcNAc transferase I. As most glycosylation reactions arenot complete, a particular glycosylated protein generally contains arange of different carbohydrate structures (i.e. glycoforms) on itssurface. Thus, the mere presence of trace amounts (i.e., less than 5%)of a particular structure like Man₅GlcNAc₂ is of little practicalrelevance for producing mammalian- or human-like glycoproteins. It isthe formation of a GlcNAc transferase I-accepting Man₅GlcNAc₂intermediate (FIG. 1B) in high yield (i.e., above 30%), which isrequired. The formation of this intermediate is necessary to enablesubsequent in vivo synthesis of complex N-glycans on glycosylatedproteins of interest (target proteins).

[0116] Accordingly, some or all of the Man₅GlcNAc₂ produced by theselected host cell must be a productive substrate for enzyme activitiesalong a mammalian glycosylation pathway, e.g., can serve as a substratefor a GlcNAc transferase I activity in vivo, thereby forming thehuman-like N-glycan intermediate GlcNAcMan₅GlcNAc₂ in the host cell. Ina preferred embodiment, at least 10%, more preferably at least 30% andmost preferably 50% or more of the Man₅GlcNAc₂ intermediate produced inthe host cell of the invention is a productive substrate for GnTI invivo. It is understood that if, for example, GlcNAcMan₅GlcNAc₂ isproduced at 10% and Man₅GlcNAc₂ is produced at 25% on a target protein,that the total amount of transiently produced Man₅GlcNAc₂ is 35% becauseGlcNAcMan₅GlcNAc₂ is a product of Man₅GlcNAc₂.

[0117] One of ordinary skill in the art can select host cells fromnature, e.g., existing fungi or other lower eukaryotes that producesignificant levels of Man₅GlcNAc₂ in vivo. As yet, however, no lowereukaryote has been shown to provide such structures in vivo in excess of1.8% of the total N-glycans (see e.g. Maras et al., 1997).Alternatively, such host cells may be genetically engineered to producethe Man₅GlcNAc₂ structure in vivo. Methods such as those described inU.S. Pat. No. 5,595,900 may be used to identify the absence or presenceof particular glycosyltransferases, mannosidases and sugar nucleotidetransporters in a target host cell or organism of interest.

Inactivation of Undesirable Host Cell Glycosylation Enzymes

[0118] The methods of the invention are directed to making host cellswhich produce glycoproteins having altered, and preferably human-like,N-glycan structures. In a preferred embodiment, the methods are directedto making host cells in which oligosaccharide precursors are enriched inMan₅GlcNAc₂. Preferably, a eukaryotic host cell is used that does notexpress one or more enzymes involved in the production of high mannosestructures. Such a host cell may be found in nature or may beengineered, e.g., starting with or derived from one of many such mutantsalready described in yeasts. Thus, depending on the selected host cell,one or a number of genes that encode enzymes known to be characteristicof non-human glycosylation reactions will have to be deleted. Such genesand their corresponding proteins have been extensively characterized ina number of lower eukaryotes (e.g., S. cerevisiae, T. reesei, A.nidulans etc.), thereby providing a list of known glycosyltransferasesin lower eukaryotes, their activities and their respective geneticsequence. These genes are likely to be selected from the group ofmannosyltransferases e.g. 1,3 mannosyltransferases (e.g. MNN1 in S.cerevisiae) (Graham, 1991), 1,2 mannosyltransferases (e.g. KTR/KREfamily from S. cerevisiae), 1,6 mannosyltransferases (OCHI from S.cerevisiae), mannosylphosphate transferases and their regulators (MNN4and MNN6 from S. cerevisiae) and additional enzymes that are involved inaberrant, i.e. non human, glycosylation reactions. Many of these geneshave in fact been deleted individually giving rise to viable phenotypeswith altered glycosylation profiles. Examples are shown in Table 1.

[0119] Preferred lower eukaryotic host cells of the invention, asdescribed herein to exemplify the required manipulation steps, arehypermannosylation-minus (och1) mutants of Pichia pastoris or K. lactis.Like other lower eukaryotes, P. pastoris processes Man₉GlcNAc₂structures in the ER with an α-1,2-mannosidase to yield Man₈GlcNAc₂(FIG. 1A). Through the action of several mannosyltransferases, thisstructure is then converted to hypermannosylated structures(Man_(>9)GlcNAc₂), also known as mannans. In addition, it has been foundthat P. pastoris is able to add non-terminal phosphate groups, throughthe action of mannosylphosphate transferases, to the carbohydratestructure. This differs from the reactions performed in mammalian cells,which involve the removal rather than addition of mannose sugars. It isof particular importance to eliminate the ability of the eukaryotic hostcell, e.g., fungus, to hypermannosylate an existing Man₈GlcNAc₂structure. This can be achieved by either selecting for a host cell thatdoes not hypermannosylate or by genetically engineering such a cell.

[0120] Genes that are involved in the hypermannosylation process havebeen identified, e.g., in Pichia pastoris, and by creating mutations inthese genes, one can reduce the production of “undesirable” glycoforms.Such genes can be identified by homology to existingmannosyltransferases or their regulators (e.g., OCH1, MNN4, MNN6, MNN1)found in other lower eukaryotes such as C. albicans, Pichia angusta orS. cerevisiae or by mutagenizing the host strain and selecting for aglycosylation phenotype with reduced mannosylation. Based on homologiesamongst known mannosyltransferases and mannosylphosphate transferases,one may either design PCR primers (examples of which are shown in Table2), or use genes or gene fragments encoding such enzymes as probes toidentify homologs in DNA libraries of the target or a related organism.Alternatively, one may identify a functional homolog havingmannosyltransferase activity by its ability to complement particularglycosylation phenotypes in related organisms. TABLE 2 PCR PrimersTarget Gene(s) PCR primer A PCR primer B in P. pastoris HomologsATGGCGAAGGCAGA TTAGTCCTTCCAAC 1,6- OCH1 S. cerevisiae, TGGCAGT TTCCTTCmannosyltransferase Pichia albicans TAYTGGMGNGTNGA GCRTCNCCCCANCK 1,2KTR/KRE family, RCYNGAYATHAA YTCRTA mannosyltransferases S. cerevisiae

[0121] To obtain the gene or genes encoding 1,6-mannosyltransferaseactivity in P. pastoris, for example, one would carry out the followingsteps: OCHI mutants of S. cerevisiae are temperature sensitive and areslow growers at elevated temperatures. One can thus identify functionalhomologs of OCHI in P. pastoris by complementing an OCHI mutant of S.cerevisiae with a P. pastoris DNA or cDNA library. Mutants of S.cerevisiae are available, e.g., from Stanford University and arecommercially available from ResGen, an Invitrogen Corp. (Carlsbad,Calif.). Mutants that display a normal growth phenotype at elevatedtemperature, after having been transformed with a P. pastoris DNAlibrary, are likely to carry an OCHI homolog of P. pastoris. Such alibrary can be created by partially digesting chromosomal DNA of P.pastoris with a suitable restriction enzyme and, after inactivating therestriction enzyme, ligating the digested DNA into a suitable vector,which has been digested with a compatible restriction enzyme.

[0122] Suitable vectors include, e.g., pRS314, a low copy (CEN6/ ARS4)plasmid based on pBluescript containing the Trp1 marker (Sikorski, R.S., and Hieter, P.,1989, Genetics 122, pg 19-27) and pFL44S, a high copy(2μ) plasmid based on a modified pUC19 containing the URA3 marker(Bonneaud, N., et al., 1991, Yeast 7, pg. 609-615). Such vectors arecommonly used by academic researchers and similar vectors are availablefrom a number of different vendors (e.g., Invitrogen (Carlsbad, Calif.);Pharmacia (Piscataway, N.J.); New England Biolabs (Beverly, Mass.)).Further examples include pYES/GS, 2μ origin of replication based yeastexpression plasmid from Invitrogen, or Yep24 cloning vehicle from NewEngland Biolabs.

[0123] After ligation of the chromosomal DNA and the vector, one maytransform the DNA library into a strain of S. cerevisiae with a specificmutation and select for the correction of the corresponding phenotype.After sub-cloning and sequencing the DNA fragment that is able torestore the wild-type phenotype, one may use this fragment to eliminatethe activity of the gene product encoded by OCHI in P. pastoris using invivo mutagenesis and/or recombination techniques well-known to thoseskilled in the art.

[0124] Alternatively, if the entire genomic sequence of a particularhost cell, e.g., fungus, of interest is known, one may identify suchgenes simply by searching publicly available DNA databases, which areavailable from several sources, such as NCBI, Swissprot. For example, bysearching a given genomic sequence or database with sequences from aknown 1,6 mannosyltransferase gene (e.g., OCHI from S. cerevisiae), onecan identify genes of high homology in such a host cell genome which may(but do not necessarily) encode proteins that have1,6-mannosyltransferase activity. Nucleic acid sequence homology aloneis not enough to prove, however, that one has identified and isolated ahomolog encoding an enzyme having the same activity. To date, forexample, no data exist to show that an OCHI deletion in P. pastoriseliminates the crucial initiating 1,6-mannosyltransferase activity.(Martinet et al. Biotech. Letters 20(12) (December 1998): 1171-1177;Contreras et al. WO 02/00856 A2). Thus, no data prove that the P.pastoris OCHI gene homolog actually encodes that function. Thatdemonstration is provided for the first time herein.

[0125] Homologs to several S. cerevisiae mannosyltransferases have beenidentified in P. pastoris using these approaches. Homologous genes oftenhave similar functions to genes involved in the mannosylation ofproteins in S. cerevisiae and thus their deletion may be used tomanipulate the glycosylation pattern in P. pastoris or, by analogy, inany other host cell, e.g., fungus, plant, insect or animal cells, withsimilar glycosylation pathways.

[0126] The creation of gene knock-outs, once a given target genesequence has been determined, is a well-established technique in the artand can be carried out by one of ordinary skill in the art (see, e.g.,R. Rothstein, (1991) Methods in Enzymology, vol. 194, p. 281). Thechoice of a host organism may be influenced by the availability of goodtransformation and gene disruption techniques.

[0127] If several mannosyltransferases are to be knocked out, the methoddeveloped by Alani and Kleckner, (Genetics 116:541-545 (1987)), forexample, enables the repeated use of a selectable marker, e.g., the URA3marker in yeast, to sequentially eliminate all undesirable endogenousmannosyltransferase activity. This technique has been refined by othersbut basically involves the use of two repeated DNA sequences, flanking acounter selectable marker. For example: URA3 may be used as a marker toensure the selection of a transformants that have integrated aconstruct. By flanking the URA3 marker with direct repeats one may firstselect for transformants that have integrated the construct and havethus disrupted the target gene. After isolation of the transformants,and their characterization, one may counter select in a second round forthose that are resistant to 5-fluoroorotic acid (5-FOA). Colonies thatare able to survive on plates containing 5-FOA have lost the URA3 markeragain through a crossover event involving the repeats mentioned earlier.This approach thus allows for the repeated use of the same marker andfacilitates the disruption of multiple genes without requiringadditional markers. Similar techniques for sequential elimination ofgenes adapted for use in another eukaryotic host cells with otherselectable and counter-selectable markers may also be used.

[0128] Eliminating specific mannosyltransferases, such as 1,6mannosyltransferase (OCH1) or mannosylphosphate transferases (MNN6, orgenes complementing lbd mutants) or regulators (MNN4) in P. pastorisenables one to create engineered strains of this organism whichsynthesize primarily Man₈GlcNAc₂ and which can be used to further modifythe glycosylation pattern to resemble more complex glycoform structures,e.g., those produced in mammalian, e.g., human cells. A preferredembodiment of this method utilizes DNA sequences encoding biochemicalglycosylation activities to eliminate similar or identical biochemicalfunctions in P. pastoris to modify the glycosylation structure ofglycoproteins produced in the genetically altered P. pastoris strain.

[0129] Methods used to engineer the glycosylation pathway in yeasts asexemplified herein can be used in filamentous fungi to produce apreferred substrate for subsequent modification. Strategies formodifying glycosylation pathways in A. niger and other filamentousfungi, for example, can be developed using protocols analogous to thosedescribed herein for engineering strains to produce human-likeglycoproteins in yeast. Undesired gene activities involved in 1,2mannosyltransferase activity, e.g., KTR/KRE homologs, are modified oreliminated. A filamentous fungus, such as Aspergillus, is a preferredhost because it lacks the 1,6 mannosyltransferase activity and as such,one would not expect a hypermannosylating gene activity, e.g. OCH1, inthis host. By contrast, other desired activities (e.g.,α-1,2-mannosidase, UDP-GlcNAc transporter, glycosyltransferase (GnT),galactosyltransferase (GalT) and sialyltransferase (ST)) involved inglycosylation are introduced into the host using the targeting methodsof the invention.

Engineering or Selecting Hosts Having Diminished Initiating α-1,6Mannosyltransferase Activity

[0130] In a preferred embodiment, the method of the invention involvesmaking or using a host cell which is diminished or depleted in theactivity of an initiating α-1,6-mannosyltransferase, i.e., an initiationspecific enzyme that initiates outer chain mannosylation on the α-1,3arm of the Man₃GlcNAc₂ core structure. In S. cerevisiae, this enzyme isencoded by the OCHI gene. Disruption of the OCH1gene in S. cerevisiaeresults in a phenotype in which N-linked sugars completely lack thepoly-mannose outer chain. Previous approaches for obtainingmammalian-type glycosylation in fungal strains have requiredinactivation of OCHI (see, e.g., Chiba, 1998). Disruption of theinitiating α-1,6-mannosyltransferase activity in a host cell of theinvention may be optional, however (depending on the selected hostcell), as the Och1p enzyme requires an intact Man₈GlcNAc₂ for efficientmannose outer chain initiation. Thus, host cells selected or producedaccording to this invention which accumulate oligosaccharides havingseven or fewer mannose residues may produce hypoglycosylated N-glycansthat will likely be poor substrates for Och1p (see, e.g., Nakayama,1997).

[0131] The OCHI gene was cloned from P. pastoris (Example 1) and K.lactis (Example 16), as described. The nucleic acid and amino acidsequences of the OCHI gene from K. lactis are set forth in SEQ ID NOS:41 and 42. Using gene-specific primers, a construct was made from eachclone to delete the OCHI gene from the genome of P. pastoris and K.lactis (Examples 1 and 16, respectively). Host cells depleted ininitiating α-1,6-mannosyltransferase activity and engineered to produceN-glycans having a Man₅GlcNAc₂ carbohydrate structure were therebyobtained (see, e.g., FIGS. 5 and 6; Examples 11 and 16).

[0132] Thus, in another embodiment, the invention provides an isolatednucleic acid molecule having a nucleic acid sequence comprising orconsisting of at least forty-five, preferably at least 50, morepreferably at least 60 and most preferably 75 or more nucleotideresidues of the K. lactis OCHI gene (SEQ ID NO: 41), and homologs,variants and derivatives thereof. The invention also provides nucleicacid molecules that hybridize under stringent conditions to theabove-described nucleic acid molecules. Similarly, isolated polypeptides(including muteins, allelic variants, fragments, derivatives, andanalogs) encoded by the nucleic acid molecules of the invention areprovided. Also provided are vectors, including expression vectors, whichcomprise the above nucleic acid molecules of the invention, as describedfurther herein. Similarly, host cells transformed with the nucleic acidmolecules or vectors of the invention are provided.

Host Cells of the Invention

[0133] A preferred host cell of the invention is a lower eukaryoticcell, e.g., yeast, a unicellular and multicellular or filamentousfungus. However, a wide variety of host cells are envisioned as beinguseful in the methods of the invention. Plant cells or insect cells, forinstance, may be engineered to express a human-like glycoproteinaccording to the invention (Examples 17 and 18). Likewise, a variety ofnon-human, mammalian host cells may be altered to express morehuman-like or otherwise altered glycoproteins using the methods of theinvention. As one of skill in the art will appreciate, any eukaryotichost cell (including a human cell) may be used in conjunction with alibrary of the invention to express one or more chimeric proteins whichis targeted to a subcellular location, e.g., organelle, in the host cellwhere the activity of the protein is modified, and preferably isenhanced. Such a protein is preferably—but need not necessarily be—anenzyme involved in protein glycosylation, as exemplified herein. It isenvisioned that any protein coding sequence may be targeted and selectedfor modified activity in a eukaryotic host cell using the methodsdescribed herein.

[0134] Lower eukaryotes that are able to produce glycoproteins havingthe attached N-glycan Man₅GlcNAc₂ are particularly useful because (a)lacking a high degree of mannosylation (e.g. greater than 8 mannoses perN-glycan, or especially 30-40 mannoses), they show reducedimmunogenicity in humans; and (b) the N-glycan is a substrate forfurther glycosylation reactions to form an even more human-likeglycoform, e.g., by the action of GlcNAc transferase I (FIG. 1B; β1,2GnTI) to form GlcNAcMan₅GlcNAc₂. A yield is obtained of greater than 30mole %, more preferably a yield of 50-100 mole %, glycoproteins withN-glycans having a Man₅GlcNAc₂ structure. In a preferred embodiment,more than 50% of the Man₅GlcNAc₂ structure is shown to be a substratefor a GnTI activity and can serve as such a substrate in vivo.

[0135] Preferred lower eukaryotes of the invention include but are notlimited to: Pichia pastoris, Pichia finlandica, Pichia trehalophila,Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichiathermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi,Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomycescerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp.,Kluyveromyces lactis, Candida albicans, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Trichoderma reseei, Chrysosporiumlucknowense, Fusarium sp. Fusarium gramineum, Fusarium venenatum andNeurospora crassa.

[0136] In each above embodiment, the method is directed to making a hostcell in which the oligosaccharide precursors are enriched inMan₅GlcNAc₂. These structures are desirable because they may then beprocessed by treatment in vitro, for example, using the method of Marasand Contreras, U.S. Pat. No. 5,834,251. In a preferred embodiment,however, precursors enriched in Man₅GlcNAc₂ are processed by at leastone further glycosylation reaction in vivo—with glycosidases (e.g.,α-mannosidases) and glycosyltransferases (e.g., GnTI)—to producehuman-like N-glycans. Oligosaccharide precursors enriched inMan₅GlcNAc₂, for example, are preferably processed to those havingGlcNAcMan_(x)GlcNAc₂ core structures, wherein X is 3, 4 or 5, and ispreferably 3. N-glycans having a GlcNAcMan_(x)GlcNAc₂ core structurewhere X is greater than 3 may be converted to GlcNAcMan₃GlcNAc₂, e.g.,by treatment with an α-1,3 and/or α-1,6 mannosidase activity, whereapplicable. Additional processing of GlcNAcMan₃GlcNAc₂ by treatment withglycosyltransferases (e.g., GnTII) produces GlcNAc₂Man₃GlcNAc₂ corestructures which may then be modified, as desired, e.g., by ex vivotreatment or by heterologous expression in the host cell of additionalglycosylation enzymes, including glycosyltransferases, sugartransporters and mannosidases (see below), to become human-likeN-glycans.

[0137] Preferred human-like glycoproteins which may be producedaccording to the invention include those which comprise N-glycans havingseven or fewer, or three or fewer, mannose residues; and which compriseone or more sugars selected from the group consisting of galactose,GlcNAc, sialic acid, and fucose.

Formation of Complex N-Glycans

[0138] Formation of complex N-glycan synthesis is a sequential processby which specific sugar residues are removed and attached to the coreoligosaccharide structure. In higher eukaryotes, this is achieved byhaving the substrate sequentially exposed to various processing enzymes.These enzymes carry out specific reactions depending on their particularlocation within the entire processing cascade. This “assembly line”consists of ER, early, medial and late Golgi, and the trans Golginetwork all with their specific processing environment. To re-create theprocessing of human glycoproteins in the Golgi and ER of lowereukaryotes, numerous enzymes (e.g. glycosyltransferases, glycosidases,phosphatases and transporters) have to be expressed and specificallytargeted to these organelles, and preferably, in a location so that theyfunction most efficiently in relation to their environment as well as toother enzymes in the pathway.

[0139] Because one goal of the methods described herein is to achieve arobust protein production strain that is able to perform well in anindustrial fermentation process, the integration of multiple genes intothe host cell chromosome involves careful planning. As described above,one or more genes which encode enzymes known to be characteristic ofnon-human glycosylation reactions are preferably deleted. The engineeredcell strain is transformed with a range of different genes encodingdesired activities, and these genes are transformed in a stable fashion,thereby ensuring that the desired activity is maintained throughout thefermentation process.

[0140] Any combination of the following enzyme activities may beengineered singly or multiply into the host using methods of theinvention: sialyltransferases, mannosidases, fucosyltransferases,galactosyltransferases, GlcNAc transferases, ER and Golgi specifictransporters (e.g. syn- and antiport transporters for UDP-galactose andother precursors), other enzymes involved in the processing ofoligosaccharides, and enzymes involved in the synthesis of activatedoligosaccharide precursors such as UDP-galactose andCMP-N-acetylneuraminic acid. Preferably, enzyme activities areintroduced on one or more nucleic acid molecules (see also below).Nucleic acid molecules may be introduced singly or multiply, e.g., inthe context of a nucleic acid library such as a combinatorial library ofthe invention. It is to be understood, however, that single or multipleenzymatic activities may be introduced into a host cell in any fashion,including but not limited to protein delivery methods and/or by use ofone or more nucleic acid molecules without necessarily using a nucleicacid library or combinatorial library of the invention.

[0141] Expression of Glycosyltransferases to Produce Complex N-Glycans:

[0142] With DNA sequence information, the skilled artisan can clone DNAmolecules encoding GnT activities (e.g., Examples 3 and 4). Usingstandard techniques well-known to those of skill in the art, nucleicacid molecules encoding GnTI, II, III, IV or V (or encodingcatalytically active fragments thereof) may be inserted into appropriateexpression vectors under the transcriptional control of promoters andother expression control sequences capable of driving transcription in aselected host cell of the invention, e.g., a fungal host such as Pichiasp., Kluyveromyces sp. and Aspergillus sp., as described herein, suchthat one or more of these mammalian GnT enzymes may be activelyexpressed in a host cell of choice for production of a human-likecomplex glycoprotein (e.g., Examples 15, 17 and 18).

[0143] Several individual glycosyltransferases have been cloned andexpressed in S. cerevisiae (GAlT, GnTI), Aspergillus nidulans (GnTI) andother fungi, without however demonstrating the desired outcome of“humanization” on the glycosylation pattern of the organisms (Yoshida,1995; Schwientek, 1995; Kalsner, 1995). It was speculated that thecarbohydrate structure required to accept sugars by the action of suchglycosyltransferases was not present in sufficient amounts, which mostlikely contributed to the lack of complex N-glycan formation.

[0144] A preferred method of the invention provides the functionalexpression of a GnT, such as GnTI, in the early or medial Golgiapparatus as well as ensuring a sufficient supply of UDP-GlcNAc (e.g.,by expression of a UDP-GlcNAc transporter; see below).

[0145] Methods for Providing Sugar Nucleotide Precursors to the GolgiApparatus:

[0146] For a glycosyltransferase to function satisfactorily in theGolgi, the enzyme requires a sufficient concentration of an appropriatenucleotide sugar, which is the high-energy donor of the sugar moietyadded to a nascent glycoprotein. In humans, the full range of nucleotidesugar precursors (e.g. UDP-N-acetylglucosamine,UDP-N-acetylgalactosamine, CMP-N-acetylneuraminic acid, UDP-galactose,etc.) are generally synthesized in the cytosol and transported into theGolgi, where they are attached to the core oligosaccharide byglycosyltransferases.

[0147] To replicate this process in non-human host cells such as lowereukaryotes, sugar nucleoside specific transporters have to be expressedin the Golgi to ensure adequate levels of nucleoside sugar precursors(Sommers, 1981; Sommers, 1982; Perez, 1987). Nucleotide sugars may beprovided to the appropriate compartments, e.g., by expressing in thehost microorganism an exogenous gene encoding a sugar nucleotidetransporter. The choice of transporter enzyme is influenced by thenature of the exogenous glycosyltransferase being used. For example, aGlcNAc transferase may require a UDP-GlcNAc transporter, afucosyltransferase may require a GDP-fucose transporter, agalactosyltransferase may require a UDP-galactose transporter, and asialyltransferase may require a CMP-sialic acid transporter.

[0148] The added transporter protein conveys a nucleotide sugar from thecytosol into the Golgi apparatus, where the nucleotide sugar may bereacted by the glycosyltransferase, e.g. to elongate an N-glycan. Thereaction liberates a nucleoside diphosphate or monophosphate, e.g. UDP,GDP, or CMP. Nucleoside monophosphates can be directly exported from theGolgi in exchange for nucleoside triphosphate sugars by an antiportmechanism. Accumulation of a nucleoside diphosphate, however, inhibitsthe further activity of a glycosyltransferase. As this reaction appearsto be important for efficient glycosylation, it is frequently desirableto provide an expressed copy of a gene encoding a nucleotidediphosphatase. The diphosphatase (specific for UDP or GDP asappropriate) hydrolyzes the diphosphonucleoside to yield a nucleosidemonosphosphate and inorganic phosphate.

[0149] Suitable transporter enzymes, which are typically of mammalianorigin, are described below. Such enzymes may be engineered into aselected host cell using the methods of the invention (see also Examples7-10).

[0150] In another example, α2,3- or α2,6-sialyltransferase capsgalactose residues with sialic acid in the trans-Golgi and TGN of humansleading to a mature form of the glycoprotein (FIG. 1B). To reengineerthis processing step into a metabolically engineered yeast or funguswill require (1) α2,3- or α2,6-sialyltransferase activity and (2) asufficient supply of CMP-N-acetyl neuraminic acid, in the late Golgi ofyeast (Example 6). To obtain sufficient α2,3-sialyltransferase activityin the late Golgi, for example, the catalytic domain of a knownsialyltransferase (e.g. from humans) has to be directed to the lateGolgi in fungi (see above). Likewise, transporters have to be engineeredto allow the transport of CMP-N-acetyl neuraminic acid into the lateGolgi. There is currently no indication that fungi synthesize or caneven transport sufficient amounts of CMP-N-acetyl neuraminic acid intothe Golgi. Consequently, to ensure the adequate supply of substrate forthe corresponding glycosyltransferases, one has to metabolicallyengineer the production of CMP-sialic acid into the fungus.

[0151] UDP-N-acetylglucosamine

[0152] The cDNA of human UDP-N-acetylglucosamine transporter, which wasrecognized through a homology search in the expressed sequence tagsdatabase (dbEST), has been cloned (Ishida, 1999 J. Biochem. 126(1):68-77). The mammalian Golgi membrane transporter forUDP-N-acetylglucosamine was cloned by phenotypic correction with cDNAfrom canine kidney cells (MDCK) of a recently characterizedKluyveromyces lactis mutant deficient in Golgi transport of the abovenucleotide sugar (Guillen, 1998). Results demonstrate that the mammalianGolgi UDP-GlcNAc transporter gene has all of the necessary informationfor the protein to be expressed and targeted functionally to the Golgiapparatus of yeast and that two proteins with very different amino acidsequences may transport the same solute within the same Golgi membrane(Guillen, 1998).

[0153] Accordingly, one may incorporate the expression of a UDP-GlcNActransporter in a host cell by means of a nucleic acid construct whichmay contain, for example: (1) a region by which the transformedconstruct is maintained in the cell (e.g. origin of replication or aregion that mediates chromosomal integration), (2) a marker gene thatallows for the selection of cells that have been transformed, includingcounterselectable and recyclable markers such as ura3 or T-urf13(Soderholm, 2001) or other well characterized selection-markers (e.g.,his4, bla, Sh ble etc.), (3) a gene or fragment thereof encoding afunctional UDP-GlcNAc transporter (e.g. from K. lactis, (Abeijon, (1996)Proc. Natl. Acad. Sci. U.S.A. 93:5963-5968), or from H. sapiens (Ishida,1996), and (4) a promoter activating the expression of the abovementioned localization/catalytic domain fusion construct library.

[0154] GDP-Fucose

[0155] The rat liver Golgi membrane GDP-fucose transporter has beenidentified and purified by Puglielli, L. and C. B. Hirschberg(Puglielli, 1999 J. Biol. Chem. 274(50):35596-35600). The correspondinggene has not been identified, however, N-terminal sequencing can be usedfor the design of oligonucleotide probes specific for the correspondinggene. These oligonucleotides can be used as probes to clone the geneencoding for GDP-fucose transporter.

[0156] UDP-Galactose

[0157] Two heterologous genes, gmal2(+) encoding alpha1,2-galactosyltransferase (alpha 1,2 GalT) from Schizosaccharomycespombe and (hUGT2) encoding human UDP-galactose (UDP-Gal) transporter,have been functionally expressed in S. cerevisiae to examine theintracellular conditions required for galactosylation. Correlationbetween protein galactosylation and UDP-galactose transport activityindicated that an exogenous supply of UDP-Gal transporter, rather thanalpha 1,2 GalT played a key role for efficient galactosylation in S.cerevisiae (Kainuma, 1999 Glycobiology 9(2): 133-141). Likewise, anUDP-galactose transporter from S. pombe was cloned (Aoki, 1999 J.Biochem. 126(5): 940-950; Segawa, 1999 Febs Letters 451(3): 295-298).

[0158] CMP-N-acetylneuraminic acid (CMP-Sialic acid).

[0159] Human CMP-sialic acid transporter (hCST) has been cloned andexpressed in Lec 8 CHO cells (Aoki, 1999; Eckhardt, 1997). Thefunctional expression of the murine CMP-sialic acid transporter wasachieved in Saccharomyces cerevisiae (Berninsone, 1997). Sialic acid hasbeen found in some fungi, however it is not clear whether the chosenhost system will be able to supply sufficient levels of CMP-Sialic acid.Sialic acid can be either supplied in the medium or alternatively fungalpathways involved in sialic acid synthesis can also be integrated intothe host genome.

[0160] Expression of Diphosphatases:

[0161] When sugars are transferred onto a glycoprotein, either anucleoside diphosphate or monophosphate is released from the sugarnucleotide precursors. While monophosphates can be directly exported inexchange for nucleoside triphosphate sugars by an antiport mechanism,diphosphonucleosides (e.g. GDP) have to be cleaved by phosphatases (e.g.GDPase) to yield nucleoside monophosphates and inorganic phosphate priorto being exported. This reaction appears to be important for efficientglycosylation, as GDPase from S. cerevisiae has been found to benecessary for mannosylation. However, the enzyme only has 10% of theactivity towards UDP (Berninsone, 1994). Lower eukaryotes often do nothave UDP-specific diphosphatase activity in the Golgi as they do notutilize UDP-sugar precursors for glycoprotein synthesis in the Golgi.Schizosaccharomyces pombe, a yeast which adds galactose residues to cellwall polysaccharides (from UDP-galactose), was found to have specificUDPase activity, further suggesting the requirement for such an enzyme(Berninsone, 1994). UDP is known to be a potent inhibitor ofglycosyltransferases and the removal of this glycosylation side productis important to prevent glycosyltransferase inhibition in the lumen ofthe Golgi (Khatara et al. 1974).

[0162] Methods for Altering N-Glycans in a Host by Expressing a TargetedEnzymatic Activity from a Nucleic Acid Molecule

[0163] The present invention further provides a method for producing ahuman-like glycoprotein in a non-human host cell comprising the step ofintroducing into the cell one or more nucleic acid molecules whichencode an enzyme or enzymes for production of the Man₅GlcNAc₂carbohydrate structure. In one preferred embodiment, a nucleic acidmolecule encoding one or more mannosidase activities involved in theproduction of Man₅GlcNAc₂ from Man₈GlcNAc₂ or Man₉GlcNAc₂ is introducedinto the host. The invention additionally relates to methods for makingaltered glycoproteins in a host cell comprising the step of introducinginto the host cell a nucleic acid molecule which encodes one or moreglycosylation enzymes or activities. Preferred enzyme activities areselected from the group consisting of UDP-GlcNAc transferase,UDP-galactosyltransferase, GDP-fucosyltransferase,CMP-sialyltransferase, UDP-GlcNAc transporter, UDP-galactosetransporter, GDP-fucose transporter, CMP-sialic acid transporter, andnucleotide diphosphatases. In a particularly preferred embodiment, thehost is selected or engineered to express two or more enzymaticactivities in which the product of one activity increases substratelevels of another activity, e.g., a glycosyltransferase and acorresponding sugar transporter, e.g., GnTI and UDP-GlcNAc transporteractivities. In another preferred embodiment, the host is selected orengineered to expresses an activity to remove products which may inhibitsubsequent glycosylation reactions, e.g. a UDP- or GDP-specificdiphosphatase activity.

[0164] Preferred methods of the invention involve expressing one or moreenzymatic activities from a nucleic acid molecule in a host cell andcomprise the step of targeting at least one enzymatic activity to adesired subcellular location (e.g., an organelle) by forming a fusionprotein comprising a catalytic domain of the enzyme and a cellulartargeting signal peptide, e.g., a heterologous signal peptide which isnot normally ligated to or associated with the catalytic domain. Thefusion protein is encoded by at least one genetic construct (“fusionconstruct”) comprising a nucleic acid fragment encoding a cellulartargeting signal peptide ligated in the same translational reading frame(“in-frame”) to a nucleic acid fragment encoding an enzyme (e.g.,glycosylation enzyme), or catalytically active fragment thereof.

[0165] The targeting signal peptide component of the fusion construct orprotein is preferably derived from a member of the group consisting of:membrane-bound proteins of the ER or Golgi, retrieval signals, Type IImembrane proteins, Type I membrane proteins, membrane spanningnucleotide sugar transporters, mannosidases, sialyltransferases,glucosidases, mannosyltransferases and phosphomannosyltransferases.

[0166] The catalytic domain component of the fusion construct or proteinis preferably derived from a glycosidase, mannosidase or aglycosyltransferase activity derived from a member of the groupconsisting of GnTI, GnTII, GnTIII, GnTIV, GnTV, GnTVI, GalT,Fucosyltransferase and Sialyltransferase. The catalytic domainpreferably has a pH optimum within 1.4 pH units of the average pHoptimum of other representative enzymes in the organelle in which theenzyme is localized, or has optimal activity at a pH between 5.1 and8.0. In a preferred embodiment, the catalytic domain encodes amannosidase selected from the group consisting of C. elegans mannosidaseIA, C. elegans mannosidase IB, D. melanogaster mannosidase IA, H.sapiens mannosidase IB, P. citrinum mannosidase I, mouse mannosidase IA,mouse mannosidase IB, A. nidulans mannosidase IA, A. nidulansmannosidase IB, A. nidulans mannosidase IC, mouse mannosidase II, C.elegans mannosidase II, H. sapiens mannosidase II, and mannosidase III.

Selecting a Glycosylation Enzyme: pH Optima and Subcellular Localization

[0167] In one embodiment of the invention, a human-like glycoprotein ismade efficiently in a non-human eukaryotic host cell by introducing intoa subcellular compartment of the cell a glycosylation enzyme selected tohave a pH optimum similar to the pH optima of other enzymes in thetargeted subcellular compartment. For example, most enzymes that areactive in the ER and Golgi apparatus of S. cerevisiae have pH optimathat are between about 6.5 and 7.5 (see Table 3). Because theglycosylation of proteins is a highly evolved and efficient process, theinternal pH of the ER and the Golgi is likely also in the range of about6-8. All previous approaches to reduce mannosylation by the action ofrecombinant mannosidases in fungal hosts, however, have introducedenzymes that have a pH optimum of around pH 5.0 (Martinet et al., 1998,and Chiba et al., 1998). At pH 7.0, the in vitro determined activity ofthose mannosidases is reduced to less than 10%, which is likelyinsufficient activity at their point of use, namely, the ER and earlyGolgi, for the efficient in vivo production of Man₅GlcNAc₂ on N-glycans.

[0168] Accordingly, a preferred embodiment of this invention targets aselected glycosylation enzyme (or catalytic domain thereof), e.g., anα-mannosidase, to a subcellular location in the host cell (e.g., anorganelle) where the pH optimum of the enzyme or domain is within 1.4 pHunits of the average pH optimum of other representative marker enzymeslocalized in the same organelle(s). The pH optimum of the enzyme to betargeted to a specific organelle should be matched with the pH optimumof other enzymes found in the same organelle to maximize the activityper unit enzyme obtained. Table 3 summarizes the activity ofmannosidases from various sources and their respective pH optima. Table4 summarizes their typical subcellular locations. TABLE 3 Mannosidasesand their pH optimum. pH Source Enzyme optimum Reference Aspergillusα-1,2-mannosidase 5.0 Ichishima et al., saitoi 1999 Biochem. J. 339(Pt3): 589-597 Trichoderma α-1,2-mannosidase 5.0 Maras et al., 2000 J.reesei Biotechnol. 77(2-3): 255-263 Penicillium α-D-1,2-mannosidase 5.0Yoshida et al., citrinum 1993 Biochem. J. 290(Pt 2): 349-354 C. elegansα-1,2-mannosidase 5.5 see FIG. 11 Aspergillus α-1,2-mannosidase 6.0Eades and Hintz, nidulans 2000 Homo sapiens α-1,2-mannosidase 6.0 IA(Golgi) Homo sapiens α-1,2-mannosidase 6.0 IB (Golgi) Lepidopteran TypeI α-1,2-Man₆- 6.0 Ren et al., 1995 insect cells mannosidase Biochem.34(8): 2489-2495 Homo sapiens α-D-mannosidase 6.0 Chandrasekaran et al.,1984 Cancer Res. 44(9): 4059-68 Xanthomonas α-1,2,3-mannosidase 6.0 U.S.Pat. No. manihotis 6,300,113 Mouse IB α-1,2-mannosidase 6.5 Schneikertand (Golgi) Herscovics, 1994 Glycobiology. 4(4): 445-50 Bacillus sp.α-D-1,2-mannosidase 7.0 Maruyama et al., (secreted) 1994 CarbohydrateRes. 251: 89-98

[0169] In a preferred embodiment, a particular enzyme or catalyticdomain is targeted to a subcellular location in the host cell by meansof a chimeric fusion construct encoding a protein comprising a cellulartargeting signal peptide not normally associated with the enzymaticdomain. Preferably, an enzyme or domain is targeted to the ER, theearly, medial or late Golgi of the trans Golgi apparatus of the hostcell.

[0170] In a more preferred embodiment, the targeted glycosylation enzymeis a mannosidase, glycosyltransferase or a glycosidase. In an especiallypreferred embodiment, mannosidase activity is targeted to the ER or cisGolgi, where the early reactions of glycosylation occur. While thismethod is useful for producing a human-like glycoprotein in a non-humanhost cell, it will be appreciated that the method is also useful moregenerally for modifying carbohydrate profiles of a glycoprotein in anyeukaryotic host cell, including human host cells.

[0171] Targeting sequences which mediate retention of proteins incertain organelles of the host cell secretory pathway are well-known anddescribed in the scientific literature and public databases, asdiscussed in more detail below with respect to libraries for selectionof targeting sequences and targeted enzymes. Such subcellular targetingsequences may be used alone or in combination to target a selectedglycosylation enzyme (or catalytic domain thereof) to a particularsubcellular location in a host cell, i.e., especially to one where theenzyme will have enhanced or optimal activity based on pH optima or thepresence of other stimulatory factors.

[0172] When one attempts to trim high mannose structures to yieldMan₅GlcNAc₂ in the ER or the Golgi apparatus of a host cell such as S.cerevisiae, for example, one may choose any enzyme or combination ofenzymes that (1) has a sufficiently close pH optimum (i.e. between pH5.2 and pH 7.8), and (2) is known to generate, alone or in concert, thespecific isomeric Man₅GlcNAc₂ structure required to accept subsequentaddition of GlcNAc by GnTI. Any enzyme or combination of enzymes that isshown to generate a structure that can be converted to GlcNAcMan₅GlcNAc₂by GnTI in vitro would constitute an appropriate choice. This knowledgemay be obtained from the scientific literature or experimentally.

[0173] For example, one may determine whether a potential mannosidasecan convert Man₈GlcNAc₂-2AB (2-aminobenzamide) to Man₅GlcNAc₂-AB andthen verify that the obtained Man₅GlcNAc₂-2AB structure can serve asubstrate for GnTI and UDP-GlcNAc to give GlcNAcMan₅GlcNAc₂ in vitro.Mannosidase IA from a human or murine source, for example, would be anappropriate choice (see, e.g., Example 11). Examples described hereinutilize 2-aminobenzamide labeled N-linked oligomannose followed by HPLCanalysis to make this determination. TABLE 4 Cellular location and pHoptima of various glycosylation-related enzymes of S. cerevisiae. pHGene Activity Location optimum Reference(s) KTR1 α-1,2 Golgi 7.0 Romeroet al., mannosyltransferase 1997 Biochem. J. 321(Pt 2): 289- 295 MNS1α-1,2-mannosidase ER 6.5 CWH41 glucosidase I ER 6.8 —mannosyltransferase Golgi 7-8 Lehele and Tanner, 1974 Biochim. Biophys.Acta 350(1): 225-235 KRE2 α-1,2 Golgi 6.5-9.0 Romero et al.,mannosyltransferase 1997

[0174] Accordingly, a glycosylation enzyme such as an α-1,2-mannosidaseenzyme used according to the invention has an optimal activity at a pHof between 5.1 and 8.0. In a preferred embodiment, the enzyme has anoptimal activity at a pH of between 5.5 and 7.5. The C. elegansmannosidase enzyme, for example, works well in the methods of theinvention and has an apparent pH optimum of about 5.5). Preferredmannosidases include those listed in Table 3 having appropriate pHoptima, e.g. Aspergillus nidulans, Homo sapiens IA (Golgi), Homo sapiensIB (Golgi), Lepidopteran insect cells (IPLB-SF21AE), Homo sapiens, mouseIB (Golgi), Xanthomonas manihotis, Drosophila melanogaster and C.elegans.

[0175] The experiment which illustrates the pH optimum for anα-1,2-mannosidase enzyme is described in Example 14. A chimeric fusionprotein BB27-2 (Saccharomyces MAN10 (s)/C. elegans mannosidase IB Δ31),which leaks into the medium was subjected to various pH ranges todetermine the optimal activity of the enzyme. The results of theexperiment show that the α-1,2-mannosidase has an optimal pH of about5.5 for its function (FIG. 11).

[0176] In a preferred embodiment, a single cloned mannosidase gene isexpressed in the host organism. However, in some cases it may bedesirable to express several different mannosidase genes, or severalcopies of one particular gene, in order to achieve adequate productionof Man₅GlcNAc₂. In cases where multiple genes are used, the encodedmannosidases preferably all have pH optima within the preferred range ofabout 5.1 to about 8.0, or especially between about 5.5 and about 7.5.Preferred mannosidase activities include α-1,2-mannosidases derived frommouse, human, Lepidoptera, Aspergillus nidulans, or Bacillus sp., C.elegans, D. melanogaster, P. citrinum, X. laevis or A. nidulans.

In Vivo Alteration of Host Cell Glycosylation Using a Combinatorial DNALibrary

[0177] Certain methods of the invention are preferably (but need notnecessarily be) carried out using one or more nucleic acid libraries. Anexemplary feature of a combinatorial nucleic acid library of theinvention is that it comprises sequences encoding cellular targetingsignal peptides and sequences encoding proteins to be targeted (e.g.,enzymes or catalytic domains thereof, including but not limited to thosewhich mediate glycosylation).

[0178] In one embodiment, a combinatorial nucleic acid librarycomprises: (a) at least two nucleic acid sequences encoding differentcellular targeting signal peptides; and (b) at least one nucleic acidsequence encoding a polypeptide to be targeted. In another embodiment, acombinatorial nucleic acid library comprises: (a) at least one nucleicacid sequence encoding a cellular targeting signal peptide; and (b) atleast two nucleic acid sequences encoding a polypeptide to be targetedinto a host cell. As described further below, a nucleic acid sequencederived from (a) and a nucleic acid sequence derived from (b) areligated to produce one or more fusion constructs encoding a cellulartargeting signal peptide functionally linked to a polypeptide domain ofinterest. One example of a functional linkage is when the cellulartargeting signal peptide is ligated to the polypeptide domain ofinterest in the same translational reading frame (“in-frame”).

[0179] In a preferred embodiment, a combinatorial DNA library expressesone or more fusion proteins comprising cellular targeting signalpeptides ligated in-frame to catalytic enzyme domains. The encodedfusion protein preferably comprises a catalytic domain of an enzymeinvolved in mammalian- or human-like modification of N-glycans. In amore preferred embodiment, the catalytic domain is derived from anenzyme selected from the group consisting of mannosidases,glycosyltransferases and other glycosidases which is ligated in-frame toone or more targeting signal peptides. The enzyme domain may beexogenous and/or endogenous to the host cell. A particularly preferredsignal peptide is one normally associated with a protein that undergoesER to Golgi transport.

[0180] The combinatorial DNA library of the present invention may beused for producing and localizing in vivo enzymes involved in mammalian-or human-like N-glycan modification. The fusion constructs of thecombinatorial DNA library are engineered so that the encoded enzymes arelocalized in the ER, Golgi or the trans-Golgi network of the host cellwhere they are involved in producing particular N-glycans on aglycoprotein of interest. Localization of N-glycan modifying enzymes ofthe present invention is achieved through an anchoring mechanism orthrough protein-protein interaction where the localization peptideconstructed from the combinatorial DNA library localizes to a desiredorganelle of the secretory pathway such as the ER, Golgi or the transGolgi network.

[0181] An example of a useful N-glycan, which is produced efficientlyand in sufficient quantities for further modification by human-like(complex) glycosylation reactions is Man₅GlcNAc₂. A sufficient amount ofMan₅GlcNAc₂ is needed on a glycoprotein of interest for furtherhuman-like processing in vivo (e.g., more than 30 mole %). TheMan₅GlcNAc₂ intermediate may be used as a substrate for further N-glycanmodification to produce GlcNAcMan₅GlcNAc₂ (FIG. 1B; see above).Accordingly, the combinatorial DNA library of the present invention maybe used to produce enzymes which subsequently produce GlcNAcMan₅GlcNAc₂,or other desired complex N-glycans, in a useful quantity.

[0182] A further aspect of the fusion constructs produced using thecombinatorial DNA library of the present invention is that they enablesufficient and often near complete intracellular N-glycan trimmingactivity in the engineered host cell. Preferred fusion constructsproduced by the combinatorial DNA library of the invention encode aglycosylation enzyme, e.g., a mannosidase, which is effectivelylocalized to an intracellular host cell compartment and thereby exhibitsvery little and preferably no extracellular activity. The preferredfusion constructs of the present invention that encode a mannosidaseenzyme are shown to localize where the N-glycans are modified, namely,the ER and the Golgi. The fusion enzymes of the present invention aretargeted to such particular organelles in the secretory pathway wherethey localize and act upon N-glycans such as Man₈GlcNAc₂ to produceMan₅GlcNAc₂ on a glycoprotein of interest.

[0183] Enzymes produced by the combinatorial DNA library of the presentinvention can modify N-glycans on a glycoprotein of interest as shownfor K3 or IFN-β proteins expressed in P. pastoris, as shown in FIG. 5and FIG. 6, respectively (see also Examples 2 and 11). It is, however,appreciated that other types of glycoproteins, without limitation,including erythropoietin, cytokines such as interferon-α, interferon-β,interferon-γ, interferon-ω, and granulocyte-CSF, coagulation factorssuch as factor VIII, factor IX, and human protein C, soluble IgEreceptor α-chain, IgG, IgG fragments, IgM, urokinase, chymase, and ureatrypsin inhibitor, IGF-binding protein, epidermal growth factor, growthhormone-releasing factor, annexin V fusion protein, angiostatin,vascular endothelial growth factor-2, mycloid progenitor inhibitoryfactor-1, osteoprotegerin, α-1 antitrypsin, DNase II and α-feto proteinsmay be glycosylated in this way.

[0184] Constructing a Combinatorial DNA Library of Fusion Constructs:

[0185] A combinatorial DNA library of fusion constructs features one ormore cellular targeting signal peptides (“targeting peptides”) generallyderived from N-terminal domains of native proteins (e.g., by makingC-terminal deletions). Some targeting peptides, however, are derivedfrom the C-terminus of native proteins (e.g. SEC12). Membrane-boundproteins of the ER or the Golgi are preferably used as a source fortargeting peptide sequences. These proteins have sequences encoding acytosolic tail (ct), a transmembrane domain (tmd) and a stem region (sr)which are varied in length. These regions are recognizable by proteinsequence alignments and comparisons with known homologs and/or otherlocalized proteins (e.g., comparing hydrophobicity plots).

[0186] The targeting peptides are indicated herein as short (s), medium(m) and long (l) relative to the parts of a type II membrane. Thetargeting peptide sequence indicated as short (s) corresponds to thetransmembrane domain (tmd) of the membrane-bound protein. The targetingpeptide sequence indicated as long (l) corresponds to the length of thetransmembrane domain (tmd) and the stem region (sr). The targetingpeptide sequence indicated as medium (m) corresponds to thetransmembrane domain (tmd) and approximately half the length of the stemregion (sr). The catalytic domain regions are indicated herein by thenumber of nucleotide deletion with respect to its wild-typeglycosylation enzyme.

[0187] Sub-Libraries

[0188] In some cases a combinatorial nucleic acid library of theinvention may be assembled directly from existing or wild-type genes. Ina preferred embodiment, the DNA library is assembled from the fusion oftwo or more sub-libraries. By the in-frame ligation of thesub-libraries, it is possible to create a large number of novel geneticconstructs encoding useful targeted protein domains such as those whichhave glycosylation activities.

[0189] Catalytic Domain Sub-Libraries Encoding Glycosylation Activities

[0190] One useful sub-library includes DNA sequences encoding enzymessuch as glycosidases (e.g., mannosidases), glycosyltransferases (e.g.,fucosyltransferases, galactosyltransferases, glucosyltransferases),GlcNAc transferases and sialyltransferases. Catalytic domains may beselected from the host to be engineered, as well as from other relatedor unrelated organisms. Mammalian, plant, insect, reptile, algal orfungal enzymes are all useful and should be chosen to represent a broadspectrum of biochemical properties with respect to temperature and pHoptima. In a preferred embodiment, genes are truncated to give fragmentssome of which encode the catalytic domains of the enzymes. By removingendogenous targeting sequences, the enzymes may then be redirected andexpressed in other cellular loci.

[0191] The choice of such catalytic domains may be guided by theknowledge of the particular environment in which the catalytic domain issubsequently to be active. For example, if a particular glycosylationenzyme is to be active in the late Golgi, and all known enzymes of thehost organism in the late Golgi have a certain pH optimum, or the lateGolgi is known to have a particular pH, then a catalytic domain ischosen which exhibits adequate, and preferably maximum, activity at thatpH, as discussed above.

[0192] Targeting Peptide Sequence Sub-Libraries

[0193] Another useful sub-library includes nucleic acid sequencesencoding targeting signal peptides that result in localization of aprotein to a particular location within the ER, Golgi, or trans Golginetwork. These targeting peptides may be selected from the host organismto be engineered as well as from other related or unrelated organisms.Generally such sequences fall into three categories: (1) N-terminalsequences encoding a cytosolic tail (ct), a transmembrane domain (tmd)and part or all of a stem region (sr), which together or individuallyanchor proteins to the inner (lumenal) membrane of the Golgi; (2)retrieval signals which are generally found at the C-terminus such asthe HDEL or KDEL tetrapeptide; and (3) membrane spanning regions fromvarious proteins, e.g., nucleotide sugar transporters, which are knownto localize in the Golgi.

[0194] In the first case, where the targeting peptide consists ofvarious elements (ct, tmd and sr), the library is designed such that thect, the tmd and various parts of the stem region are represented.Accordingly, a preferred embodiment of the sub-library of targetingpeptide sequences includes ct, tmd, and/or sr sequences frommembrane-bound proteins of the ER or Golgi. In some cases it may bedesirable to provide the sub-library with varying lengths of srsequence. This may be accomplished by PCR using primers that bind to the5′ end of the DNA encoding the cytosolic region and employing a seriesof opposing primers that bind to various parts of the stem region.

[0195] Still other useful sources of targeting peptide sequences includeretrieval signal peptides, e.g. the tetrapeptides HDEL or KDEL, whichare typically found at the C-terminus of proteins that are transportedretrograde into the ER or Golgi. Still other sources of targetingpeptide sequences include (a) type II membrane proteins, (b) the enzymeslisted in Table 3, (c) membrane spanning nucleotide sugar transportersthat are localized in the Golgi, and (d) sequences referenced in Table5. TABLE 5 Sources of useful compartmental targeting sequences Gene orLocation of Sequence Organism Function Gene Product MNSI A. nidulansα-1,2-mannosidase ER MNSI A. niger α-1,2-mannosidase ER MNSI S.cerevisiae α-1,2-mannosidase ER GLSI S. cerevisiae glucosidase ER GLSIA. niger glucosidase ER GLSI A. nidulans glucosidase ER HDEL atUniversal in retrieval signal ER C-terminus fungi SEC12 S. cerevisiaeCOPII vesicle protein ER/Golgi SEC12 A. niger COPII vesicle proteinER/Golgi OCH1 S. cerevisiae 1,6-mannosyltransferase Golgi (cis) OCH1 P.pastoris 1,6-mannosyltransferase Golgi (cis) MNN9 S. cerevisiae1,6-mannosyltransferase Golgi complex MNN9 A. niger undetermined GolgiVAN1 S. cerevisiae undetermined Golgi VAN1 A. niger undetermined GolgiANP1 S. cerevisiae undetermined Golgi HOCI S. cerevisiae undeterminedGolgi MNN10 S. cerevisiae undetermined Golgi MNN10 A. niger undeterminedGolgi MNN11 S. cerevisiae undetermined Golgi (cis) MNN11 A. nigerundetermined Golgi (cis) MNT1 S. cerevisiae 1,2-mannosyltransferaseGolgi (cis, medial KTR1 P. pastoris undetermined Golgi (medial) KRE2 P.pastoris undetermined Golgi (medial) KTR3 P. pastoris undetermined Golgi(medial) MNN2 S. cerevisiae 1,2-mannosyltransferase Golgi (medial) KTR1S. cerevisiae undetermined Golgi (medial) KTR2 S. cerevisiaeundetermined Golgi (medial) MNN1 S. cerevisiae 1,3-mannosyltransferaseGolgi (trans) MNN6 S. cerevisiae Phosphomannosyl- Golgi (trans)transferase 2,6 ST H. sapiens 2,6-sialyltransferase trans Golgi networkUDP-Gal T S. pombe UDP-Gal transporter Golgi

[0196] In any case, it is highly preferred that targeting peptidesequences are selected which are appropriate for the particularenzymatic activity or activities to function optimally within thesequence of desired glycosylation reactions. For example, in developinga modified microorganism capable of terminal sialylation of nascentN-glycans, a process which occurs in the late Golgi in humans, it isdesirable to utilize a sub-library of targeting peptide sequencesderived from late Golgi proteins. Similarly, the trimming of Man₈GlcNAc₂by an α-1,2-mannosidase to give Man₅GlcNAc₂ is an early step in complexN-glycan formation in humans (FIG. 1B). It is therefore desirable tohave this reaction occur in the ER or early Golgi of an engineered hostmicroorganism. A sub-library encoding ER and early Golgi retentionsignals is used.

[0197] A series of fusion protein constructs (i.e., a combinatorial DNAlibrary) is then constructed by functionally linking one or a series oftargeting peptide sequences to one or a series of sequences encodingcatalytic domains. In a preferred embodiment, this is accomplished bythe in-frame ligation of a sub-library comprising DNA encoding targetingpeptide sequences (above) with a sub-library comprising DNA encodingglycosylation enzymes or catalytically active fragments thereof (seebelow).

[0198] The resulting library comprises synthetic genes encodingtargeting peptide sequence-containing fusion proteins. In some cases itis desirable to provide a targeting peptide sequence at the N-terminusof a fusion protein, or in other cases at the C-terminus. In some cases,targeting peptide sequences may be inserted within the open readingframe of an enzyme, provided the protein structure of individual foldeddomains is not disrupted. Each type of fusion protein is constructed (ina step-wise directed or semi-random fashion) and optimal constructs maybe selected upon transformation of host cells and characterization ofglycosylation patterns in transformed cells using methods of theinvention.

[0199] Generating Additional Sequence Diversity

[0200] The method of this embodiment is most effective when a nucleicacid, e.g., a DNA library transformed into the host contains a largediversity of sequences, thereby increasing the probability that at leastone transformant will exhibit the desired phenotype. Single amino acidmutations, for example, may drastically alter the activity ofglycoprotein processing enzymes (Romero et al., 2000). Accordingly,prior to transformation, a DNA library or a constituent sub-library maybe subjected to one or more techniques to generate additional sequencediversity. For example, one or more rounds of gene shuffling, errorprone PCR, in vitro mutagenesis or other methods for generating sequencediversity, may be performed to obtain a larger diversity of sequenceswithin the pool of fusion constructs.

[0201] Expression Control Sequences

[0202] In addition to the open reading frame sequences described above,it is generally preferable to provide each library construct withexpression control sequences, such as promoters, transcriptionterminators, enhancers, ribosome binding sites, and other functionalsequences as may be necessary to ensure effective transcription andtranslation of the fusion proteins upon transformation of fusionconstructs into the host organism.

[0203] Suitable vector components, e.g., selectable markers, expressioncontrol sequences (e.g., promoter, enhancers, terminators and the like)and, optionally, sequences required for autonomous replication in a hostcell, are selected as a function of which particular host cell ischosen. Selection criteria for suitable vector components for use in aparticular mammalian or a lower eukaryotic host cell are routine.Preferred lower eukaryotic host cells of the invention include Pichiapastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae,Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichiamethanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp.,Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candidaalbicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp. Fusariumgramineum, Fusarium venenatum and Neurospora crassa. Where the host isPichia pastoris, suitable promoters include, for example, the AOX1,AOX2, GAPDH and P40 promoters.

[0204] Selectable Markers

[0205] It is also preferable to provide each construct with at least oneselectable marker, such as a gene to impart drug resistance or tocomplement a host metabolic lesion. The presence of the marker is usefulin the subsequent selection of transformants; for example, in yeast theURA3, HIS4, SUC2, G418, BLA, or SH BLE genes may be used. A multitude ofselectable markers are known and available for use in yeast, fungi,plant, insect, mammalian and other eukaryotic host cells.

[0206] Transformation

[0207] The nucleic acid library is then transformed into the hostorganism. In yeast, any convenient method of DNA transfer may be used,such as electroporation, the lithium chloride method, or the spheroplastmethod. In filamentous fungi and plant cells, conventional methodsinclude particle bombardment, electroporation and agrobacterium mediatedtransformation. To produce a stable strain suitable for high-densityculture (e.g., fermentation in yeast), it is desirable to integrate theDNA library constructs into the host chromosome. In a preferredembodiment, integration occurs via homologous recombination, usingtechniques well-known in the art. For example, DNA library elements areprovided with flanking sequences homologous to sequences of the hostorganism. In this manner, integration occurs at a defined site in thehost genome, without disruption of desirable or essential genes.

[0208] In an especially preferred embodiment, library DNA is integratedinto the site of an undesired gene in a host chromosome, effecting thedisruption or deletion of the gene. For example, integration into thesites of the OCH1, MNN1, or MNN4 genes allows the expression of thedesired library DNA while preventing the expression of enzymes involvedin yeast hypermannosylation of glycoproteins. In other embodiments,library DNA may be introduced into the host via a nucleic acid molecule,plasmid, vector (e.g., viral or retroviral vector), chromosome, and maybe introduced as an autonomous nucleic acid molecule or by homologous orrandom integration into the host genome. In any case, it is generallydesirable to include with each library DNA construct at least oneselectable marker gene to allow ready selection of host organisms thathave been stably transformed. Recyclable marker genes such as ura3,which can be selected for or against, are especially suitable.

[0209] Screening and Selection Processes

[0210] After transformation of the host strain with the DNA library,transformants displaying a desired glycosylation phenotype are selected.Selection may be performed in a single step or by a series of phenotypicenrichment and/or depletion steps using any of a variety of assays ordetection methods. Phenotypic characterization may be carried outmanually or using automated high-throughput screening equipment.Commonly, a host microorganism displays protein N-glycans on the cellsurface, where various glycoproteins are localized.

[0211] One may screen for those cells that have the highestconcentration of terminal GlcNAc on the cell surface, for example, orfor those cells which secrete the protein with the highest terminalGlcNAc content. Such a screen may be based on a visual method, like astaining procedure, the ability to bind specific terminal GlcNAc bindingantibodies or lectins conjugated to a marker (such lectins are availablefrom E. Y. Laboratories Inc., San Mateo, Calif.), the reduced ability ofspecific lectins to bind to terminal mannose residues, the ability toincorporate a radioactively labeled sugar in vitro, altered binding todyes or charged surfaces, or may be accomplished by using a FluorescenceAssisted Cell Sorting (FACS) device in conjunction with a fluorophorelabeled lectin or antibody (Guillen, 1998).

[0212] Accordingly, intact cells may be screened for a desiredglycosylation phenotype by exposing the cells to a lectin or antibodythat binds specifically to the desired N-glycan. A wide variety ofoligosaccharide-specific lectins are available commercially (e.g., fromE Y Laboratories, San Mateo, Calif.). Alternatively, antibodies tospecific human or animal N-glycans are available commercially or may beproduced using standard techniques. An appropriate lectin or antibodymay be conjugated to a reporter molecule, such as a chromophore,fluorophore, radioisotope, or an enzyme having a chromogenic substrate(Guillen et al., 1998. Proc. Natl. Acad. Sci. USA 95(14): 7888-7892)).

[0213] Screening may then be performed using analytical methods such asspectrophotometry, fluorimetry, fluorescence activated cell sorting, orscintillation counting. In other cases, it may be necessary to analyzeisolated glycoproteins or N-glycans from transformed cells. Proteinisolation may be carried out by techniques known in the art. In apreferred embodiment, a reporter protein is secreted into the medium andpurified by affinity chromatography (e.g. Ni-affinity orglutathione-S-transferase affinity chromatography). In cases where anisolated N-glycan is preferred, an enzyme such asendo-β-N-acetylglucosaminidase (Genzyme Co., Boston, Mass.; New EnglandBiolabs, Beverly, Mass.) may be used to cleave the N-glycans fromglycoproteins. Isolated proteins or N-glycans may then be analyzed byliquid chromatography (e.g. HPLC), mass spectroscopy, or other suitablemeans. U.S. Pat. No. 5,595,900 teaches several methods by which cellswith desired extracellular carbohydrate structures may be identified. Ina preferred embodiment, MALDI-TOF mass spectrometry is used to analyzethe cleaved N-glycans.

[0214] Prior to selection of a desired transformant, it may be desirableto deplete the transformed population of cells having undesiredphenotypes. For example, when the method is used to engineer afunctional mannosidase activity into cells, the desired transformantswill have lower levels of mannose in cellular glycoprotein. Exposing thetransformed population to a lethal radioisotope of mannose in the mediumdepletes the population of transformants having the undesired phenotype,i.e. high levels of incorporated mannose (Huffaker T C and Robbins P W.,Proc Natl Acad Sci USA. 1983 December;80(24):7466-70). Alternatively, acytotoxic lectin or antibody, directed against an undesirable N-glycan,may be used to deplete a transformed population of undesired phenotypes(e.g., Stanley P and Siminovitch L. Somatic Cell Genet 1977July;3(4):391-405). U.S. Pat. No. 5,595,900 teaches several methods bywhich cells with a desired extracellular carbohydrate structures may beidentified. Repeatedly carrying out this strategy allows for thesequential engineering of more and more complex glycans in lowereukaryotes.

[0215] To detect host cells having on their surface a high degree of thehuman-like N-glycan intermediate GlcNAcMan₃GlcNAc₂, for example, one mayselect for transformants that allow for the most efficient transfer ofGlcNAc by GlcNAc Transferase from UDP-GlcNAc in an in vitro cell assay.This screen may be carried out by growing cells harboring thetransformed library under selective pressure on an agar plate andtransferring individual colonies into a 96-well microtiter plate. Aftergrowing the cells, the cells are centrifuged, the cells resuspended inbuffer, and after addition of UDP-GlcNAc and GnT II, the release of UDPis determined either by HPLC or an enzyme linked assay for UDP.Alternatively, one may use radioactively labeled UDP-GlcNAc and GnT II,wash the cells and then look for the release of radioactive GlcNAc byN-actylglucosaminidase. All this may be carried manually or automatedthrough the use of high throughput screening equipment. Transformantsthat release more UDP, in the first assay, or more radioactively labeledGlcNAc in the second assay, are expected to have a higher degree ofGlcNAcMan₃GlcNAc₂ on their surface and thus constitute the desiredphenotype. Similar assays may be adapted to look at the N-glycans onsecreted proteins as well.

[0216] Alternatively, one may use any other suitable screen such as alectin binding assay that is able to reveal altered glycosylationpatterns on the surface of transformed cells. In this case the reducedbinding of lectins specific to terminal mannoses may be a suitableselection tool. Galantus nivalis lectin binds specifically to terminalα-1,3 mannose, which is expected to be reduced if sufficient mannosidaseII activity is present in the Golgi. One may also enrich for desiredtransformants by carrying out a chromatographic separation step thatallows for the removal of cells containing a high terminal mannosecontent. This separation step would be carried out with a lectin columnthat specifically binds cells with a high terminal mannose content(e.g., Galantus nivalis lectin bound to agarose, Sigma, St.Louis, Mo.)over those that have a low terminal mannose content.

[0217] In addition, one may directly create such fusion proteinconstructs, as additional information on the localization of activecarbohydrate modifying enzymes in different lower eukaryotic hostsbecomes available in the scientific literature. For example, it is knownthat human β1,4-GalTr can be fused to the membrane domain of MNT, amannosyltransferase from S. cerevisiae, and localized to the Golgiapparatus while retaining its catalytic activity (Schwientek et al.,1995). If S. cerevisiae or a related organism is the host to beengineered one may directly incorporate such findings into the overallstrategy to obtain complex N-glycans from such a host. Several such genefragments in P. pastoris have been identified that are related toglycosyltransferases in S. cerevisiae and thus could be used for thatpurpose.

[0218] Alteration of Host Cell Glycosylation Using Fusion Constructsfrom Combinatorial Libraries

[0219] The construction of a preferred combinatorial DNA library isillustrated schematically in FIG. 2 and described in Example 11. Thefusion construct may be operably linked to a multitude of vectors, suchas expression vectors well-known in the art. A wide variety of suchfusion constructs were assembled using representative activities asshown in Table 6. Combinations of targeting peptide/catalytic domainsmay be assembled for use in targeting mannosidase, glycosyltransferaseand glycosidase activities in the ER, Golgi and the trans Golgi networkaccording to the invention. Surprisingly, the same catalytic domain mayhave no effect to a very profound effect on N-glycosylation patterns,depending on the type of targeting peptide used (see, e.g., Table 7,Example 11).

[0220] Mannosidase Fusion Constructs

[0221] A representative example of a mannosidase fusion constructderived from a combinatorial DNA library of the invention is pFB8, whicha truncated Saccharomyces SEC12(m) targeting peptide (988-1296nucleotides of SEC12 from SwissProt P11655) ligated in-frame to a 187N-terminal amino acid deletion of a mouse α-mannosidase IA (Genbank AN6678787). The nomenclature used herein, thus, refers to the targetingpeptide/catalytic domain region of a glycosylation enzyme asSaccharomyces SEC12 (m)/mouse mannosidase IA Δ187. The encoded fusionprotein localizes in the ER by means of the SEC12 targeting peptidesequence while retaining its mannosidase catalytic domain activity andis capable of producing in vivo N-glycans having a Man₅GlcNAc₂ structure(Example 11; FIG. 6F, FIG. 7B).

[0222] The fusion construct pGC5, Saccharomyces MNS1(m)/mousemannosidase IB Δ99, is another example of a fusion construct havingintracellular mannosidase trimming activity (Example 11; FIG. 5D, FIG.8B). Fusion construct pBC18-5 (Saccharomyces VAN1(s)/C. elegansmannosidase IB Δ80) is yet another example of an efficient fusionconstruct capable of producing N-glycans having a Man₅GlcNAc₂ structurein vivo. By creating a combinatorial DNA library of these and other suchmannosidase fusion constructs according to the invention, a skilledartisan may distinguish and select those constructs having optimalintracellular trimming activity from those having relatively low or noactivity. Methods using combinatorial DNA libraries of the invention areadvantageous because only a select few mannosidase fusion constructs mayproduce a particularly desired N-glycan in vivo.

[0223] In addition, mannosidase trimming activity may be specific to aparticular protein of interest. Thus, it is to be further understoodthat not all targeting peptide/mannosidase catalytic domain fusionconstructs may function equally well to produce the proper glycosylationon a glycoprotein of interest. Accordingly, a protein of interest may beintroduced into a host cell transfected with a combinatorial DNA libraryto identify one or more fusion constructs which express a mannosidaseactivity optimal for the protein of interest. One skilled in the artwill be able to produce and select optimal fusion construct(s) using thecombinatorial DNA library approach described herein.

[0224] It is apparent, moreover, that other such fusion constructsexhibiting localized active mannosidase catalytic domains (or moregenerally, domains of any enzyme) may be made using techniques such asthose exemplified in Example 11 and described herein. It will be amatter of routine experimentation for one skilled in the art to make anduse the combinatorial DNA library of the present invention to optimize,for example, Man₅GlcNAc₂ production from a library of fusion constructsin a particular expression vector introduced into a particular hostcell.

[0225] Glycosyltransferase Fusion Constructs

[0226] Similarly, a glycosyltransferase combinatorial DNA library wasmade using the methods of the invention. A combinatorial DNA library ofsequences derived from glycosyltransferase I (GnTI) activities wereassembled with targeting peptides and screened for efficient productionin a lower eukaryotic host cell of a GlcNAcMan₅GlcNAc₂ N-glyeanstructure on a marker glycoprotein. A fusion construct shown to produceGlcNAcMan₅GlcNAc₂ (pPB104), Saccharomyces MNN9(s)/human GnTI Δ38 wasidentified (Example 15). A wide variety of such GnTI fusion constructswere assembled (Example 15, Table 10). Other combinations of targetingpeptide/GnTI catalytic domains can readily be assembled by making acombinatorial DNA library. It is also apparent to one skilled in the artthat other such fusion constructs exhibiting glycosyltransferaseactivity may be made as demonstrated in Example 15. It will be a matterof routine experimentation for one skilled in the art to use thecombinatorial DNA library method described herein to optimizeGlcNAcMan₅GlcNAc₂ production using a selected fusion construct in aparticular expression vector and host cell line.

[0227] As stated above for mannosidase fusion constructs, not alltargeting peptide/GnTI catalytic domain fusion constructs will functionequally well to produce the proper glycosylation on a glycoprotein ofinterest as described herein. However, one skilled in the art will beable to produce and select optimal fusion construct(s) using a DNAlibrary approach as described herein. Example 15 illustrates a preferredembodiment of a combinatorial DNA library comprising targeting peptidesand GnTI catalytic domain fusion constructs involved in producingglycoproteins with predominantly GlcNAcMan₅GlcNAc₂ structure.

[0228] Using Multiple Fusion Constructs to Alter Host Cell Glycosylation

[0229] In another example of using the methods and libraries of theinvention to alter host cell glycosylation, a P. pastoris strain with anOCHI deletion that expresses a reporter protein (K3) was transformedwith multiple fusion constructs isolated from combinatorial libraries ofthe invention to convert high mannose N-glycans to human-like N-glycans(Example 15). First, the mannosidase fusion construct pFB8(Saccharomyces SEC12 (m)/mouse mannosidase IA Δ187) was transformed intoa P. pastoris strain lacking 1,6 initiating mannosyltransferasesactivity (i.e. och1 deletion; Example 1). Second, pPB103 comprising a K.lactis MNN2-2 gene (Genbank AN AF106080) encoding an UDP-GlcNActransporter was constructed to increase further production ofGlcNAcMan₅GlcNAc₂. The addition of the UDP-GlcNAc transporter increasedproduction of GlcNAcMan₅GlcNAc₂ significantly in the P. pastoris strainas illustrated in FIG. 10B. Third, pPB104 comprising Saccharomyces MNN9(s)/human GnTI Δ38 was introduced into the strain. This P. pastorisstrain is referred to as “PBP-3.”

[0230] It is understood by one skilled in the art that host cells suchas the above-described yeast strains can be sequentially transformedand/or co-transformed with one or more expression vectors. It is alsounderstood that the order of transformation is not particularly relevantin producing the glycoprotein of interest. The skilled artisanrecognizes the routine modifications of the procedures disclosed hereinmay provide improved results in the production of the glycoprotein ofinterest.

[0231] The importance of using a particular targeting peptide sequencewith a particular catalytic domain sequence becomes readily apparentfrom the experiments described herein. The combinatorial DNA libraryprovides a tool for constructing enzyme fusions that are involved inmodifying N-glycans on a glycoprotein of interest, which is especiallyuseful in producing human-like glycoproteins. (Any enzyme fusion,however, may be selected using libraries and methods of the invention.)Desired transformants expressing appropriately targeted, activeα-1,2-mannosidase produce K3 with N-glycans of the structure Man₅GlcNAc₂as shown in FIGS. 5D and 5E. This confers a reduced molecular mass tothe cleaved glycan compared to the K3 of the parent OCHI deletionstrain, as was detected by MALDI-TOF mass spectrometry in FIG. 5C.

[0232] Similarly, the same approach was used to produce another secretedglycoprotein: IFN-β comprising predominantly Man₅GlcNAc₂. TheMan₅GlcNAc₂ was removed by PNGase digestion (Papac et al. 1998Glycobiology 8, 445-454) and subjected to MALDI-TOF as shown in FIG.6A-6F. A single prominent peak at 1254 (m/z) confirms Man₅GlcNA₂production on IFN-β in FIGS. 6E (pGC5) (Saccharomyces MNS1(m)/mousemannosidase IB Δ99) and 6F (pFB8) (Saccharomyces SEC12 (m)/mousemannosidase IA Δ187). Furthermore, in the P. pastoris strain PBP-3comprising pFB8 (Saccharomyces SEC12 (m)/mouse mannosidase IA Δ187),pPB104 (Saccharomyces MNN9 (s)/human GnTI Δ38) and pPB103 (K. lactisMNN2-2 gene), the hybrid N-glycan GlcNAcMan₅GlcNAc₂ [b] was detected byMALDI-TOF (FIG. 10).

[0233] After identifying transformants with a high degree of mannosetrimming, additional experiments were performed to confirm thatmannosidase (trimming) activity occurred in vivo and was notpredominantly the result of extracellular activity in the growth medium(Example 13; FIGS. 7-9).

[0234] Host Cells

[0235] Although the present invention is exemplified using a P. pastorishost organism, it is understood by those skilled in the art that othereukaryotic host cells, including other species of yeast and fungalhosts, may be altered as described herein to produce human-likeglycoproteins. The techniques described herein for identification anddisruption of undesirable host cell glycosylation genes, e.g. OCH1, isunderstood to be applicable for these and/or other homologous orfunctionally related genes in other eukaryotic host cells such as otheryeast and fungal strains. As described in Example 16, och1 mnn1 geneswere deleted from K. lactis to engineer a host cell leading to N-glycansthat are completely converted to Man₅GlcNAc₂ by 1,2-mannosidase (FIG.12C).

[0236] The MNN1 gene was cloned from K. lactis as described in Example16. The nucleic acid and deduced amino acid sequences of the K. lactisMNN1 gene are shown in SEQ ID NOS: 43 and 44, respectively. Usinggene-specific primers, a construct was made to delete the MNN1 gene fromthe genome of K. lactis (Example 16). Host cells depleted in och1 andmnn1 activities produce N-glycans having a Man₉GlcNAc₂ carbohydratestructure (see, e.g., FIG. 10). Such host cells may be engineeredfurther using, e.g., methods and libraries of the invention, to producemammalian- or human-like glycoproteins.

[0237] Thus, in another embodiment, the invention provides an isolatednucleic acid molecule having a nucleic acid sequence comprising orconsisting of at least forty-five, preferably at least 50, morepreferably at least 60 and most preferably 75 or more nucleotideresidues of the K. lactis MNN1 gene (SEQ ID NO: 43), and homologs,variants and derivatives thereof. The invention also provides nucleicacid molecules that hybridize under stringent conditions to theabove-described nucleic acid molecules. Similarly, isolated polypeptides(including muteins, allelic variants, fragments, derivatives, andanalogs) encoded by the nucleic acid molecules of the invention areprovided. In addition, also provided are vectors, including expressionvectors, which comprise a nucleic acid molecule of the invention, asdescribed further herein. Similarly host cells transformed with thenucleic acid molecules or vectors of the invention are provided.

[0238] Another aspect of the present invention thus relates to anon-human eukaryotic host strain expressing glycoproteins comprisingmodified N-glycans that resemble those made by human-cells. Performingthe methods of the invention in species other than yeast and fungalcells is thus contemplated and encompassed by this invention. It iscontemplated that a combinatorial nucleic acid library of the presentinvention may be used to select constructs that modify the glycosylationpathway in any eukaryotic host cell system. For example, thecombinatorial libraries of the invention may also be used in plants,algae and insects, and in other eukaryotic host cells, includingmammalian and human cells, to localize proteins, including glycosylationenzymes or catalytic domains thereof, in a desired location along a hostcell secretory pathway. Preferably, glycosylation enzymes or catalyticdomains and the like are targeted to a subcellular location along thehost cell secretory pathway where they are capable of functioning, andpreferably, where they are designed or selected to function mostefficiently.

[0239] As described in Examples 17 and 18, plant and insect cells may beengineered to alter the glycosylation of expressed proteins using thecombinatorial library and methods of the invention. Furthermore,glycosylation in mammalian cells, including human cells, may also bemodified using the combinatorial library and methods of the invention.It may be possible, for example, to optimize a particular enzymaticactivity or to otherwise modify the relative proportions of variousN-glycans made in a mammalian host cell using the combinatorial libraryand methods of the invention.

[0240] Examples of modifications to glycosylation which can be affectedusing a method according to this embodiment of the invention are: (1)engineering a eukaryotic host cell to trim mannose residues fromMan₈GlcNAc₂ to yield a Man₅GlcNAc₂ N-glycan; (2) engineering eukaryotichost cell to add an N-acetylglucosamine (GlcNAc) residue to Man₅GlcNAc₂by action of GlcNAc transferase I; (3) engineering a eukaryotic hostcell to functionally express an enzyme such as an N-acetylglucosaminylTransferase (GnTI, GnTII, GnTIII, GnTIV, GnTV, GnTVI), mannosidase II,fucosyltransferase (FT), galactosyl tranferase (GalT) or asialyltransferase (ST).

[0241] By repeating the method, increasingly complex glycosylationpathways can be engineered into a target host, such as a lowereukaryotic microorganism. In one preferred embodiment, the host organismis transformed two or more times with DNA libraries including sequencesencoding glycosylation activities. Selection of desired phenotypes maybe performed after each round of transformation or alternatively afterseveral transformations have occurred. Complex glycosylation pathwayscan be rapidly engineered in this manner.

[0242] Sequential Glycosylation Reactions

[0243] In a preferred embodiment, such targeting peptide/catalyticdomain libraries are designed to incorporate existing information on thesequential nature of glycosylation reactions in higher eukaryotes.Reactions known to occur early in the course of glycoprotein processingrequire the targeting of enzymes that catalyze such reactions to anearly part of the Golgi or the ER. For example, the trimming ofMan₈GlcNAc₂ to Man₅GlcNAc₂ by mannosidases is an early step in complexN-glycan formation. Because protein processing is initiated in the ERand then proceeds through the early, medial and late Golgi, it isdesirable to have this reaction occur in the ER or early Golgi. Whendesigning a library for mannosidase I localization, for example, onethus attempts to match ER and early Golgi targeting signals with thecatalytic domain of mannosidase I.

[0244] Integration Sites

[0245] As one ultimate goal of this genetic engineering effort is arobust protein production strain that is able to perform well in anindustrial fermentation process, the integration of multiple genes intothe host (e.g., fungal) chromosome preferably involves careful planning.The engineered strain may likely have to be transformed with a range ofdifferent genes, and these genes will have to be transformed in a stablefashion to ensure that the desired activity is maintained throughout thefermentation process. As described herein, any combination of variousdesired enzyme activities may be engineered into the fungal proteinexpression host, e.g., sialyltransferases, mannosidases,fucosyltransferases, galactosyltransferases, glucosyltransferases,GlcNAc transferases, ER and Golgi specific transporters (e.g. syn andantiport transporters for UDP-galactose and other precursors), otherenzymes involved in the processing of oligosaccharides, and enzymesinvolved in the synthesis of activated oligosaccharide precursors suchas UDP-galactose, CMP-N-acetylneuraminic acid. Examples of preferredmethods for modifying glycosylation in a lower eukaryotic host cell,such as Pichia pastoris, are shown in Table 6. TABLE 6 Some preferredembodiments for modifying glycosylation in a lower eukaroyticmicroorganism Suitable Suitable Suitable Suitable Sources of GeneTransporters Catalytic Localization De- and/or Desired StructureActivities Sequences letions Phosphatases Man₅GlcNAc₂ α-1,2- Mns1 (N-OCH1 none manno- terminus, S. MNN4 sidase cerevisiae) MNN6 (murine, Och1(N- human, terminus, S. Bacillus cerevisiae, sp., P. pastoris) A. Ktr1nidulans) Mnn9 Mnt1 (S. cerevisiae) KDEL, HDEL (C-terminus)GlcNAcMan₅GlcNAc₂ GlcNAc Och1 OCH1 UDP- Trans- (N-terminus, MNN4 GlcNAcferase I, S. cerevisiae, MNN6 transporter (human, P. pastoris) (human,murine, KTR1 murine, rat etc.) (N-terminus) K. lactis) Mnn1 (N- UDPaseterminus, S. (human) cerevisiae) Mnt1 (N- terminus, S. cerevisiae)GDPase (N- terminus, S. cerevisiae) GlcNAcMan₃GlcNAc₂ manno- Ktr1 OCH1UDP- sidase II Mnn1 (N- MNN4 GlcNAc terminus, S. MNN6 transportercerevisiae) (human, Mnt1 (N- murine, terminus, S. K. lactis) cerevisiae)UDPase Kre2/Mnt1 (human) (S. cerevisiae) Kre2 (P. pastoris) Ktr1 (S.cerevisiae) Ktr1 (P. pastoris) Mnn1 (S. cerevisiae) GlcNAc₍₂₋ GlcNAcMnn1 (N- OCH1 UDP- ₄₎Man₃GlcNAc₂ Trans- terminus, S. MNN4 GlcNAc feraseII, cerevisiae) MNN6 transporter III, IV, V Mnt1 (N- (human, (human,terminus, S. murine, murine) cerevisiae) K. lactis) Kre2/Mnt1 UDPase (S.(human) cerevisiae) Kre2 (P. pastoris) Ktr1 (S. cerevisiae) Ktr1 (P.pastoris) Mnn1 (S. cerevisiae) Gal₍₁₋₄₎GlcNAc₍₂₋₄₎- β-1,4- Mnn1 (N- OCH1UDP- Man₃GlcNAc₂ Galactosyl terminus, S. MNN4 Galactose transferasecerevisiae) MNN6 transporter (human) Mnt1 (N- (human, terminus, S. S.pombe) cerevisiae) Kre2/Mnt1 (S. cerevisiae) Kre2 (P. pastoris) Ktr1 (S.cerevisiae) Ktr1 (P. pastoris) Mnn1 (S. cerevisiae) NANA₍₁₋₄₎- α-2,6-KTR1 OCH1 CMP-Sialic Gal₍₁₋₄₎GlcNAc₍₂₋₄₎- Sialyl- MNN1 (N- MNN4 acidMan₃GlcNAc₂ transferase terminus, S. MNN6 transporter (human)cerevisiae) (human) α-2,3- MNT1 (N- Sialyl- terminus, S. transferasecerevisiae) Kre2/Mnt1 (S. cerevisiae) Kre2 (P. pastoris) Ktr1 (S.cerevisiae) Ktr1 (P. pastoris) MNN1 (S. cerevisiae)

[0246] As any strategy to engineer the formation of complex N-glycansinto a host cell such as a lower eukaryote involves both the eliminationas well as the addition of particular glycosyltransferase activities, acomprehensive scheme will attempt to coordinate both requirements. Genesthat encode enzymes that are undesirable serve as potential integrationsites for genes that are desirable. For example, 1,6 mannosyltransferaseactivity is a hallmark of glycosylation in many known lower eukaryotes.The gene encoding alpha-1,6 mannosyltransferase (OCH1) has been clonedfrom S. cerevisiae and mutations in the gene give rise to a viablephenotype with reduced mannosylation. The gene locus encoding alpha-1,6mannosyltransferase activity therefore is a prime target for theintegration of genes encoding glycosyltransferase activity. In a similarmanner, one can choose a range of other chromosomal integration sitesthat, based on a gene disruption event in that locus, are expected to:(1) improve the cells ability to glycosylate in a more 15 human-likefashion, (2) improve the cells ability to secrete proteins, (3) reduceproteolysis of foreign proteins and (4) improve other characteristics ofthe process that facilitate purification or the fermentation processitself.

Target Glycoproteins

[0247] The methods described herein are useful for producingglycoproteins, especially glycoproteins used therapeutically in humans.Glycoproteins having specific glycoforms may be especially useful, forexample, in the targeting of therapeutic proteins. For example,mannose-6-phosphate has been shown to direct proteins to the lysosome,which may be essential for the proper function of several enzymesrelated to lysosomal storage disorders such as Gaucher's, Hunter's,Hurler's, Scheie's, Fabry's and Tay-Sachs disease, to mention just afew. Likewise, the addition of one or more sialic acid residues to aglycan side chain may increase the lifetime of a therapeuticglycoprotein in vivo after administration. Accordingly, host cells(e.g., lower eukaryotic or mammalian) may be genetically engineered toincrease the extent of terminal sialic acid in glycoproteins expressedin the cells. Alternatively, sialic acid may be conjugated to theprotein of interest in vitro prior to administration using a sialic acidtransferase and an appropriate substrate. Changes in growth mediumcomposition may be employed in addition to the expression of enzymeactivities involved in human-like glycosylation to produce glycoproteinsmore closely resembling human forms (S. Weikert, et al., NatureBiotechnology, 1999, 17, 1116-1121; Werner, Noe, et al 1998Arzneimittelforschung 48(8):870-880; Weikert, Papac et al., 1999;Andersen and Goochee 1994 Cur. Opin. Biotechnol. 5: 546-549; Yang andButler 2000 Biotechnol. Bioengin. 68(4): 370-380). Specific glycanmodifications to monoclonal antibodies (e.g. the addition of a bisectingGlcNAc) have been shown to improve antibody dependent cell cytotoxicity(Umana P., et al. 1999), which may be desirable for the production ofantibodies or other therapeutic proteins.

[0248] Therapeutic proteins are typically administered by injection,orally, pulmonary, or other means. Examples of suitable targetglycoproteins which may be produced according to the invention include,without limitation: erythropoietin, cytokines such as interferon-α,interferon-β, interferon-γ, interferon-ω, and granulocyte-CSF,coagulation factors such as factor VIII, factor IX, and human protein C,soluble IgE receptor α-chain, IgG, IgG fragments, IgM, interleukins,urokinase, chymase, and urea trypsin inhibitor, IGF-binding protein,epidermal growth factor, growth hormone-releasing factor, annexin Vfusion protein, angiostatin, vascular endothelial growth factor-2,myeloid progenitor inhibitory factor-1, osteoprotegerin, α-1-antitrypsinand α-feto proteins.

[0249] The following are examples which illustrate the compositions andmethods of this invention. These examples should not be construed aslimiting: the examples are included for the purposes of illustrationonly.

EXAMPLE 1 Cloning and Disruption of the OCHI Gene in P. pastoria

[0250] Generation of an OCHI Mutant of P. pastoris:

[0251] A 1215 bp ORF of the P. pastoris OCHI gene encoding a putativeα-1,6 mannosyltransferase was amplified from P. pastoris genomic DNA(strain X-33, Invitrogen, Carlsbad, Calif.) using the oligonucleotides5′-ATGGCGAAGGCAGATGGCAGT-3′ (SEQ ID NO:7) and5′-TTAGTCCTTCCAACTTCCTTC-3′ (SEQ ID NO:8) which were designed based onthe P. pastoris OCHI sequence (Japanese Patent Application PublicationNo. 8-336387). Subsequently, 2685 bp upstream and 1175 bp downstream ofthe ORF of the OCHI gene were amplified from a P. pastoris genomic DNAlibrary (Boehm, T. et al. Yeast 1999 May; 15(7):563-72) using theinternal oligonucleotides 5′-ACTGCCATCTGCCTTCGCCAT-3′ (SEQ ID NO:9) inthe OCHI gene, and 5′-GTAATACGACTCACTATAGGGC-3′ T7 (SEQ ID NO:10) and5′-AATTAACCCTCACTAAAGGG-3′ T3 (SEQ ID NO:11) oligonucleotides in thebackbone of the library bearing plasmid lambda ZAP II (Stratagene, LaJolla, Calif.). The resulting 5075 bp fragment was cloned into thepCR2.1-TOPO vector (Invitrogen, Carlsbad, Calif.) and designated pBK9.

[0252] After assembling a gene knockout construct that substituted theOCH1reading frame with a HIS4 resistance gene, P. pastoris wastransformed and colonies were screened for temperature sensitivity at37° C. OCHI mutants of S. cerevisiae are temperature sensitive and areslow growers at elevated temperatures. One can thus identify functionalhomologs of OCHI in P. pastoris by complementing an OCHI mutant of S.cerevisiae with a P. pastoris DNA or cDNA library. About 20 temperaturesensitive strains were further subjected to a colony PCR screen toidentify colonies with a deleted och1 gene. Several och1 deletions wereobtained.

[0253] The linearized pBK9.1, which has 2.1 kb upstream sequence and 1.5kb down stream sequence of OCHI gene cassette carrying Pichia HIS4 gene,was transformed into P. pastoris BK1 [GS115 (his4 Invitrogen Corp., SanDiego, Calif.) carrying the human IFN-β gene in the AOX1 locus] to knockout the wild-type OCHI gene. The initial screening of transformants wasperformed using histidine drop-out medium followed by replica plating toselect the temperature sensitive colonies. Twenty out of two hundredhistidine-positive colonies showed a temperature sensitive phenotype at37° C. To exclude random integration of pBK9.1 into the Pichia genome,the 20 temperature-sensitive isolates were subjected to colony PCR usingprimers specific to the upstream sequence of the integration site and toHIS4 ORF. Two out of twenty colonies were och1 defective and furtheranalyzed using a Southern blot and a Western blot indicating thefunctional och1 disruption by the och1 knock-out construct. Genomic DNAwere digested using two separate restriction enzymes BglII and ClaI toconfirm the och1 knock-out and to confirm integration at the openreading frame. The Western Blot showed och1 mutants lacking a discreteband produced in the GS115 wild type at 46.2 kDa.

EXAMPLE 2 Engineering of P. pastoris with α-1,2-Mannosidase to ProduceMan₅GlcNAc₂-Containing IFN-β Precursors

[0254] An α-1,2-mannosidase is required for the trimming of Man₈GlcNAc₂to yield Man₅GlcNAc₂, an essential intermediate for complex N-glycanformation. While the production of a Man₅GlcNAc₂ precursor is essential,it is not necessarily sufficient for the production of hybrid andcomplex glycans because the specific isomer of Man₅GlcNAc₂ may or maynot be a substrate for GnTI. An och1 mutant of P. pastoris is engineeredto express secreted human interferon-β under the control of an aoxpromoter. A DNA library is constructed by the in-frame ligation of thecatalytic domain of human mannosidase IB (an α-1,2-mannosidase) with asub-library including sequences encoding early Golgi and ER localizationpeptides. The DNA library is then transformed into the host organism,resulting in a genetically mixed population wherein individualtransformants each express interferon-β as well as a syntheticmannosidase gene from the library. Individual transformant colonies arecultured and the production of interferon is induced by addition ofmethanol. Under these conditions, over 90% of the secreted protein isglycosylated interferon-β.

[0255] Supernatants are purified to remove salts and low-molecularweight contaminants by C₁₈ silica reversed-phase chromatography. Desiredtransformants expressing appropriately targeted, activeα-1,2-mannosidase produce interferon-β including N-glycans of thestructure Man₅GlcNAc₂, which has a reduced molecular mass compared tothe interferon-β of the parent strain. The purified interferon-β, isanalyzed by MALDI-TOF mass spectroscopy and colonies expressing thedesired form of interferon-β are identified.

EXAMPLE 3 Generation of an och1 Mutant Strain Expressing anα-1,2-Mannosidase, GnTI and GnTII for Production of a Human-LikeGlycoprotein

[0256] The 1215 bp open reading frame of the P. pastoris OCHI gene aswell as 2685 bp upstream and 1175 bp downstream was amplified by PCR(see also WO 02/00879), cloned into the pCR2.1-TOPO vector (Invitrogen)and designated pBK9. To create an och1 knockout strain containingmultiple auxotrophic markers, 100 μg of pJN329, a plasmid containing anoch1:: URA3 mutant allele flanked with SfiI restriction sites wasdigested with SfiI and used to transform P. pastoris strain JC308(Cereghino et al. Gene 263 (2001) 159-169) by electroporation. Followingincubation on defined medium lacking uracil for 10 days at roomtemperature, 1000 colonies were picked and re-streaked. URA⁺ clones thatwere unable to grow at 37° C., but grew at room temperature, weresubjected to colony PCR to test for the correct integration of theoch1::URA3 mutant allele. One clone that exhibited the expected PCRpattern was designated YJN153. The Kringle 3 domain of human plasminogen(K3) was used as a model protein. A Neo^(R) marked plasmid containingthe K3 gene was transformed into strain YJN153 and a resulting strain,expressing K3, was named BK64-1.

[0257] Plasmid pPB 103, containing the Kluyveromyces lactis MNN2-2 genewhich encodes a Golgi UDP-N-acetylglucosamine transporter wasconstructed by cloning a blunt BglII-HindIII fragment from vector pDL02(Abeijon et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:5963-5968) intoBglII and BamHI digested and blunt ended pBLADE-SX containing the P.pastoris ADE1 gene (Cereghino et al. (2001) Gene 263:159-169). Thisplasmid was linearized with EcoNI and transformed into strain BK64-1 byelectroporation and one strain confirmed to contain the MNN2-2 by PCRanalysis was named PBP 1.

[0258] A library of mannosidase constructs was generated, comprisingin-frame fusions of the leader domains of several type I or type IImembrane proteins from S. cerevisiae and P. pastoris fused with thecatalytic domains of several α-1,2-mannosidase genes from human, mouse,fly, worm and yeast sources (see, e.g., WO02/00879, incorporated hereinby reference). This library was created in a P. pastoris HIS4integration vector and screened by linearizing with SalI, transformingby electroporation into strain PBP1, and analyzing the glycans releasedfrom the K3 reporter protein. One active construct chosen was a chimeraof the 988-1296 nucleotides (C-terminus) of the yeast SEC12 gene fusedwith a N-terminal deletion of the mouse α-1,2-mannosidase IA gene (FIG.3), which was missing the 187 nucleotides. A P. pastoris strainexpressing this construct was named PBP2.

[0259] A library of GnTI constructs was generated, comprising in-framefusions of the same leader library with the catalytic domains of GnTIgenes from human, worm, frog and fly sources (WO 02/00879). This librarywas created in a P. pastoris ARG4 integration vector and screened bylinearizing with AatII, transforming by electroporation into strainPBP2, and analyzing the glycans released from K3. One active constructchosen was a chimera of the first 120 bp of the S. cerevisiae MNN9 genefused to a deletion of the human GnTI gene, which was missing the first154 bp. A P. pastoris strain expressing this construct was named PBP3.

[0260] A library of GnTII constructs was generated, which comprisedin-frame fusions of the leader library with the catalytic domains ofGnTII genes from human and rat sources (WO 02/00879). This library wascreated in a P. pastoris integration vector containing the NST^(R) geneconferring resistance to the drug nourseothricin. The library plasmidswere linearized with EcoRI, transformed into strain RDP27 byelectroporation, and the resulting strains were screened by analysis ofthe released glycans from purified K3.

[0261] Materials for the Following Reactions

[0262] MOPS, sodium cacodylate, manganese chloride, UDP-galactose andCMP-N-acetylneuraminic acid were from Sigma. Trifluoroacetic acid (TFA)was from Sigma/Aldrich, Saint Louis, Mo. Recombinant ratα2,6-sialyltransferase from Spodoptera frugiperda andβ1,4-galactosyltransferase from bovine milk were from Calbiochem (SanDiego, Calif.). Protein N-glycosidase F, mannosidases, andoligosaccharides were from Glyko (San Rafael, Calif.). DEAE ToyoPearlresin was from TosoHaas. Metal chelating “HisBind” resin was fromNovagen (Madison, Wis.). 96-well lysate-clearing plates were fromPromega (Madison, Wis.). Protein-binding 96-well plates were fromMillipore (Bedford, Mass.). Salts and buffering agents were from Sigma(St. Louis, Mo.). MALDI matrices were from Aldrich (Milwaukee, Wis.).

[0263] Protein Purification

[0264] Kringle 3 was purified using a 96-well format on a Beckman BioMek2000 sample-handling robot (Beckman/Coulter Ranch Cucamonga, Calif.).Kringle 3 was purified from expression media using a C-terminalhexa-histidine tag. The robotic purification is an adaptation of theprotocol provided by Novagen for their HisBind resin. Briefly, a 150 uL(μL) settled volume of resin is poured into the wells of a 96-welllysate-binding plate, washed with 3 volumes of water and charged with 5volumes of 50 mM NiSO4 and washed with 3 volumes of binding buffer (5 mMimidazole, 0.5M NaCl, 20 mM Tris-HCL pH7.9). The protein expressionmedia is diluted 3:2, media/PBS (60 mM PO4, 16 mM KCl, 822 mM NaClpH7.4) and loaded onto the columns. After draining, the columns arewashed with 10 volumes of binding buffer and 6 volumes of wash buffer(30 mM imidazole, 0.5M NaCl, 20 mM Tris-HCl pH7.9) and the protein iseluted with 6 volumes of elution buffer (1M imidazole, 0.5M NaCl, 20 mMTris-HCl pH7.9). The eluted glycoproteins are evaporated to dryness bylyophilyzation.

[0265] Release of N-Linked Glycans

[0266] The glycans are released and separated from the glycoproteins bya modification of a previously reported method (Papac, et al. A. J. S.(1998) Glycobiology 8, 445-454). The wells of a 96-well Multi Screen IP(Immobilon-P membrane) plate (Millipore) are wetted with 100 uL ofmethanol, washed with 3×150 uL of water and 50 uL of RCM buffer (8Murea, 360 mM Tris, 3.2 mM EDTA pH8.6), draining with gentle vacuum aftereach addition. The dried protein samples are dissolved in 30 uL of RCMbuffer and transferred to the wells containing 10 uL of RCM buffer. Thewells are drained and washed twice with RCM buffer. The proteins arereduced by addition of 60 uL of 0.1M DTT in RCM buffer for 1 hr at 37°C. The wells are washed three times with 300 uL of water andcarboxymethylated by addition of 60 uL of 0.1M iodoacetic acid for 30min in the dark at room temperature. The wells are again washed threetimes with water and the membranes blocked by the addition of 100 uL of1% PVP 360 in water for 1 hr at room temperature. The wells are drainedand washed three times with 300 uL of water and deglycosylated by theaddition of 30 uL of 10 mM NH₄HCO₃ pH 8.3 containing one milliunit ofN-glycanase (Glyko). After 16 hours at 37° C., the solution containingthe glycans was removed by centrifugation and evaporated to dryness.

[0267] Matrix Assisted Laser Desorption Ionization Time of Flight MassSpectrometry

[0268] Molecular weights of the glycans were determined using a VoyagerDE PRO linear MALDI-TOF (Applied Biosciences) mass spectrometer usingdelayed extraction. The dried glycans from each well were dissolved in15 uL of water and 0.5 uL spotted on stainless steel sample plates andmixed with 0.5 uL of S-DHB matrix (9 mg/mL of dihydroxybenzoic acid, 1mg/mL of 5-methoxysalicilic acid in 1:1 water/acetonitrile 0.1% TFA) andallowed to dry.

[0269] Ions were generated by irradiation with a pulsed nitrogen laser(337 nm) with a 4 ns pulse time. The instrument was operated in thedelayed extraction mode with a 125 ns delay and an accelerating voltageof 20 kV. The grid voltage was 93.00%, guide wire voltage was 0.10%, theinternal pressure was less than 5×10-7 torr, and the low mass gate was875 Da. Spectra were generated from the sum of 100-200 laser pulses andacquired with a 2 GHz digitizer. Man₅GlcNAc₂ oligosaccharide was used asan external molecular weight standard. All spectra were generated withthe instrument in the positive ion mode. The estimated mass accuracy ofthe spectra was 0.5%.

EXAMPLE 4 Engineering a Strain to Produce Galactosyltransferase

[0270] Galactosyltransferase Reaction

[0271] Approximately 2 mg of protein (r-K3:hPg [PBP6-5]) was purified bynickel-affinity chromatography, extensively dialyzed against 0.1% TFA,and lyophilized to dryness. The protein was redissolved in 150 μL of 50mM MOPS, 20 mM MnCl2, pH7.4. After addition of 32.5 μg (533 nmol) ofUDP-galactose and 4 mU of β 1,4-galactosyltransferase, the sample wasincubated at 37° C. for 18 hours. The samples were then dialyzed against0.1% TFA for analysis by MALDI-TOF mass spectrometry.

[0272] The spectrum of the protein reacted with galactosyltransferaseshowed an increase in mass consistent with the addition of two galactosemoieties when compared with the spectrum of a similar protein sampleincubated without enzyme. Protein samples were next reduced,carboxymethylated and deglycosylated with PNGase F. The recoveredN-glycans were analyzed by MALDI-TOF mass spectrometry. The mass of thepredominant glycan from the galactosyltransferase reacted protein wasgreater than that of the control glycan by a mass consistent with theaddition of two galactose moieties (325.4 Da).

EXAMPLE 5 Engineering a Strain to Express Functional and ActiveMannosidase II

[0273] To generate a human-like glycoform, a microorganism is engineeredto express a mannosidase II enzyme which removes the two remainingterminal mannoses from the structure GlcNAcMan₅GlcNAc₂ (see FIG. 1B). ADNA library including sequences encoding cis and medial Golgilocalization signals is fused in-frame to a library encoding mannosidaseII catalytic domains. The host organism is a strain, e.g. a yeast, thatis deficient in hypermannosylation (e.g. an ochl mutant) and providesN-glycans having the structure GlcNAcMan₅GlcNAc₂ in the Golgi and/or ER.After transformation, organisms having the desired glycosylationphenotype are selected. An in vitro assay is used in one method. Thedesired structure GlcNAcMan₃GlcNAc₂ (but not the undesiredGlcNAcMan₅GlcNAc₂) is a substrate for the enzyme GlcNAc Transferase II(see FIG. 1B). Accordingly, single colonies may be assayed using thisenzyme in vitro in the presence of the substrate, UDP-GlcNAc. Therelease of UDP is determined either by HPLC or an enzymatic assay forUDP. Alternatively, radioactively labeled UDP-GlcNAc or MALDI-TOF may beused.

[0274] The foregoing in vitro assays are conveniently performed onindividual colonies using high-throughput screening equipment.Alternatively a lectin binding assay is used. In this case the reducedbinding of lectins specific for terminal mannoses allows the selectionof transformants having the desired phenotype. For example, Galantusnivalis lectin binds specifically to terminal α-1,3-mannose, theconcentration of which is reduced in the presence of operativelyexpressed mannosidase II activity. In one suitable method, G. nivalislectin attached to a solid agarose support (available from SigmnaChemical, St. Louis, Mo.) is used to deplete the transformed populationof cells having high levels of terminal α-1,3-mannose.

EXAMPLE 6 Engineering a Strain to Express Sialyltransferase

[0275] The enzymes (α2,3-sialyltransferase and α2,6-sialyltransferaseadd terminal sialic acid to galactose residues in nascent humanN-glycans, leading to mature glycoproteins (see “α2,3 ST; α2,6 ST inFIG. 1B). In human cells, the reactions occur in the trans Golgi or TGN.Accordingly, a DNA library is constructed by the in-frame fusion ofsequences encoding sialyltransferase catalytic domains with sequencesencoding trans Golgi or TGN localization signals (Malissard et al.Biochem Biophys Res Commun Jan. 7, 2000;267(1):169-73; Borsig et al.Biochem Biophys Res Commun May 5, 1995;210(1):14-20). The host organismis a strain, e.g. a yeast, that is deficient in hypermannosylation(e.g., an och1 mutant), which provides N-glycans having terminalgalactose residues in the late Golgi or TGN, and provides a sufficientconcentration of CMP-sialic acid in the late Golgi or TGN. Followingtransformation, transformants having the desired phenotype are selected,e.g., using a fluorescent antibody specific for N-glycans having aterminal sialic acid. In addition, the strains are engineered to producethe CMP-NANA precursors.

[0276] Sialyltransferase Reaction

[0277] After resuspending the (galactosyltransferase reacted) (Example4) proteins in 10 μL of 50 mM sodium cacodylate buffer pH6.0, 300 μg(488 nmol) of CMP-N-acetylneuraminic acid (CMP-NANA) dissolved in 15 μLof the same buffer, and 5 μL (2 mU) of recombinant α-2,6sialyltransferase were added. After incubation at 37° C. for 15 hours,an additional 200 μg of CMP-NANA and 1 mU of sialyltransferase wereadded. The protein samples were incubated for an additional 8 hours andthen dialyzed and analyzed by MALDI-TOF-MS as above. The spectrum of theglycoprotein reacted with sialyltransferase showed an increase in masswhen compared with that of the starting material (the protein aftergalactosyltransferase reaction). The N-glycans were released andanalyzed as above. The increase in mass of the two ion-adducts of thepredominant glycan was consistent with the addition of two sialic acidresidues (580 and 583 Da).

EXAMPLE 7 Engineering a Strain to Express UDP-GlcNAc Transporter

[0278] The cDNA of human Golgi UDP-GlcNAc transporter has been cloned byIshida and coworkers. (Ishida, N., et al. 1999 J. Biochem. 126(1):68-77). Guillen and coworkers have cloned the canine kidney GolgiUDP-GlcNAc transporter by phenotypic correction of a Kluyveromyceslactis mutant deficient in Golgi UDP-GlcNAc transport. (Guillen, E., etal. 1998). Thus a mammalian Golgi UDP-GlcNAc transporter gene has all ofthe necessary information for the protein to be expressed and targetedfunctionally to the Golgi apparatus of yeast. These or other clonedtransporter genes may be engineered into a host organism to provideUDP-GlcNAc substrates for efficient GnT reactions in the Golgi and/or ERof the host. FIG. 10B demonstrates the effect of a strain expressing aK. lactis UDP-GlcNAc transporter. In comparison to FIG. 10A, which lacksa UDP-GlcNAc transporter, the effect of adding a UDP-GlcNAc transportershows a dramatic increase in the production of GlcNAcMan₅GlcNAc₂.

EXAMPLE 8 Engineering a Strain to Express GDP-Fucose Transporter

[0279] The rat liver Golgi membrane GDP-fucose transporter has beenidentified and purified by Puglielli, L. and C. B. Hirschberg 1999 J.Biol. Chem. 274(50):35596-35600. The corresponding gene can beidentified using standard techniques, such as N-terminal sequencing andSouthern blotting using a degenerate DNA probe. The intact gene is thenexpressed in a host microorganism that also expresses afucosyltransferase.

EXAMPLE 9 Engineering a Strain to Express UDP-Galactose Transporter

[0280] Human UDP-galactose (UDP-Gal) transporter has been cloned andshown to be active in S. cerevisiae. (Kainuma, M., et al. 1999Glycobiology 9(2): 133-141). A second human UDP-galactose transporter(hUGT1) has been cloned and functionally expressed in Chinese HamsterOvary Cells. Aoki, K., et al. 1999 J. Biochem. 126(5): 940-950.Likewise, Segawa and coworkers have cloned a UDP-galactose transporterfrom Schizosaccharomyces pombe (Segawa, H., et al. 1999 Febs Letters451(3): 295-298). These or other sequences encoding UDP-galactosetransporter activities may be introduced into a host cell directly ormay be used as a component of a sub-library of the invention to engineera strain having increased UDP-galactose transporter activity.

EXAMPLE 10 Engineering a Strain to Express CMP-Sialic Acid Transporter

[0281] Human CMP-sialic acid transporter (hCST) has been cloned andexpressed in Lec 8 CHO cells by Aoki and coworkers (1999). Molecularcloning of the hamster CMP-sialic acid transporter has also beenachieved (Eckhardt and Gerardy Schahn 1997 Eur. J. Biochem. 248(1):187-192). The functional expression of the murine CMP-sialic acidtransporter was achieved in Saccharomyces cerevisiae by Beminsone, P.,et al. 1997 J. Biol. Chem. 272 (19):12616-12619. These or othersequences encoding CMP-sialic acid transporter activities may beintroduced into a host cell directly or may be used as a component of asub-library of the invention to engineer a strain having increasedCMP-sialic acid transporter activity.

EXAMPLE 11 Engineering of P. pastoris to Produce Man₅GlcNA₂ as thePredominant N-Glycan Structure Using a Combinatorial DNA Library

[0282] An och1 mutant of P. pastoris (see Examples 1 and 3) wasengineered to express and secrete proteins such as the kringle 3 domainof human plasminogen (K3) under the control of the inducible AOXIpromoter. The Kringle 3 domain of human plasminogen (K3) was used as amodel protein. A DNA fragment encoding the K3 was amplified using Pfuturbo polymerase (Strategene, La Jolla, Calif.) and cloned into EcoRIand XbaI sites of pPICZαA (Invitrogen, Carlsbad, Calif.), resulting in aC-terminal 6-His tag. In order to improve the N-linked glycosylationefficiency of K3 (Hayes et al. 1975 J. Arch. Biochem. Biophys. 171,651-655), Pro₄₆ was replaced with Ser₄6 using site-directed mutagenesis.The resulting plasmid was designated pBK64. The correct sequence of thePCR construct was confirmed by DNA sequencing.

[0283] A combinatorial DNA library was constructed by the in-frameligation of murine α-1,2-mannosidase IB (Genbank AN 6678787) and IA(Genbank AN 6754619) catalytic domains with a sub-library includingsequences encoding Cop II vesicle, ER, and early Golgi localizationpeptides according to Table 6. The combined DNA library was used togenerate individual fusion constructs, which were then transformed intothe K3 expressing host organism, resulting in a genetically mixedpopulation wherein individual transformants each express K3 as well as alocalization signal/mannosidase fusion gene from the library. Individualtransformants were cultured and the production of K3 was induced bytransfer to a methanol containing medium. Under these conditions, after24 hours of induction, over 90% of the protein in the medium was K3. TheK3 reporter protein was purified from the supernatant to remove saltsand low-molecular weight contaminants by Ni-affinity chromatography.Following affinity purification, the protein was desalted by sizeexclusion chromatography on a Sephadex G10 resin (Sigma, St. Louis, Mo.)and either directly subjected to MALDI-TOF analysis described below orthe N-glycans were removed by PNGase digestion as described below(Release of N-glycans) and subjected to MALDI-TOF analysis Miele et al.1997 Biotechnol. Appl. Biochem. 25, 151-157.

[0284] Following this approach, a diverse set of transformants wereobtained; some showed no modification of the N-glycans compared to theoch1 knockout strain; and others showed a high degree of mannosetrimming (FIG. 5D, SE). Desired transformants expressing appropriatelytargeted, active α-1,2-mannosidase produced K3 with N-glycans of thestructure Man₅GlcNAc₂. This confers a reduced molecular mass to theglycoprotein compared to the K3 of the parent och1 deletion strain, adifference which was readily detected by MALDI-TOF mass spectrometry(FIG. 5). Table 7 indicates the relative Man₅GlcNAc₂ production levels.TABLE 7 A representative combinatorial DNA library of localizationsequences/catalytic domains exhibiting relative levels of Man₅GlcNAc₂production. Targeting peptide sequences MNS1(s) MNS1(m) MNS1(1) SEC12(s)SEC12(m) Catalytic Domains Mouse mannosidase FB4 FB5 FB6 FB7 FB8 1A Δ187++ + − ++ ++++ Mouse mannosidase GB4 GB5 GB6 GB7 GB8 1B Δ58  ++ + + ++ +Mouse mannosidase GC4 GC5 GC6 GC7 GC8 1B Δ99  − +++ + + + Mousemannosidase GD4 GD5 GD6 GD7 GD8 1B Δ170 − − − + +

[0285] TABLE 8 Another combinatorial DNA library of localizationsequences/catalytic domains exhibiting relative levels of Man₅GlcNAc₂production. Targeting peptide sequences VAN1(s) VAN1(m) VAN1(1) MNN10(s)MNN10(m) MNN10(1) Catalytic Domains C. elegans BC18-5 BC19 BC20 BC27BC28 BC29 mannosidase 1B +++++ ++++ +++ +++++ +++++ +++ Δ80 C. elegansBB18 BB19 BB20 BB18 BB19 BB20 mannosidase 1B +++++ +++++ ++++ ++++++++++ ++++ Δ31

[0286] Targeting peptides were selected from MNS I (SwissProt P32906) inS. cerevisiae (long, medium and short) (see supra Nucleic AcidLibraries; Combinatorial DNA Library of Fusion Constructs) and SEC12(SwissProt P11655) in S. cerevisiae (988-1140 nucleotides: short) and(988-1296: medium). Although majority of the targeting peptide sequenceswere N-terminal deletions, some targeting peptide sequences, such asSEC12 were C-terminal deletions. Catalytic domains used in thisexperiment were selected from mouse mannosidase IA with a 187 amino acidN-terminal deletion; and mouse mannosidase 1B with a 58, 99 and 170amino acid deletion. The number of (+)s, as used herein, indicates therelative levels of Man₅GlcNA₂ production. The notation (−) indicates noapparent production of Man₅GlcNA₂. The notation (+) indicates less than,10% production of Man₅GlcNA₂ The notation (++) indicates about 10-20%production of Man₅GlcNA₂. The notation with (+++) indicates about 20-40%production of Man₅GlcNA₂. The notation with (++++) indicates about 50%production of Man₅GlcNA₂. The notation with (+++++) indicates greaterthan 50% production of Man₅GlcNA₂.

[0287] Table 9 shows relative amount of Man₅GlcNAc₂ on secreted K3. Sixhundred and eight (608) different strains of P. pastoris, Δoch1 weregenerated by transforming them with a single construct of acombinatorial genetic library that was generated by fusing nineteen (19)α-1,2 mannosidase catalytic domains to thirty-two (32) fungal ER, andcis-Golgi leaders. TABLE 9 Amount of Man₅GlcNAc₂ on secreted K3 (% oftotal glycans) Number of constructs (%) N.D.* 19 (3.1)  0-10% 341 (56.1)10-20% 50 (8.2) 20-40& 75 (12.3) 40-60% 72 (11.8) More than 51 (8.4)^(†)  60% Total 608 (100)

[0288] Table 7 shows two constructs pFB8 and pGC5, among others,displaying Man₅GlcNA₂. Table 8 shows a more preferred construct,pBC18-5, a S. cerevisiae VAN1(s) targeting peptide sequence (fromSwissProt 23642) ligated in-frame to a C. elegans mannosidase IB(Genbank AN CAA98114) 80 amino acid N-terminal deletion (SaccharomycesVan1(s)/C. elegans mannosidase IB Δ80). This fusion construct alsoproduces a predominant Man₅GlcNA₂ structure, as shown in FIG. 5E. Thisconstruct was shown to produce greater than 50% Man₅GlcNA₂ (+++++).

[0289] Generation of a Combinatorial Localization/Mannosidase Library:

[0290] Generating a combinatorial DNA library of α-1,2-mannosidasecatalytic domains fused to targeting peptides required the amplificationof mannosidase domains with varying lengths of N-terrninal deletionsfrom a number of organisms. To approach this goal, the full length openreading frames (ORFs) of α-1,2-mannosidases were PCR amplified fromeither cDNA or genomic DNA obtained from the following sources: Homosapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans,Aspergillus nidulans and Penicillium citrinum. In each case, DNA wasincubated in the presence of oligonucleotide primers specific for thedesired mannosidase sequence in addition to reagents required to performthe PCR reaction. For example, to amplify the ORF of the M. musculusα-1,2-mannosidase IA, the 5′-primer ATGCCCGTGGGGGGCCTGTTGCCGCTCTTCAGTAGC(SEQ ID NO:12) and the 3′-primerTCATTTCTCTTTGCCATCAATTTCCTTCTTCTGTTCACGG (SEQ ID NO:13) were incubatedin the presence of Pfu DNA polymerase (Stratagene, La Jolla, Calif.) andamplified under the conditions recommended by Stratagene using thecycling parameters: 94° C. for 1 min (1 cycle); 94° C. for 30 sec, 68°C. for 30 sec, 72° C. for 3min (30 cycles). Following amplification theDNA sequence encoding the ORF was incubated at 72 ° C. for 5 min with 1UTaq DNA polymerase (Promega, Madison, Wis.) prior to ligation intopCR2.1-TOPO (Invitrogen, Carlsbad, Calif.) and transformed into TOP10chemically competent E. coli, as recomrnended by Invitrogen. The clonedPCR product was confirmed by ABI sequencing using primers specific forthe mannosidase ORF.

[0291] To generate the desired N-terminal truncations of eachmannosidase, the complete ORF of each mannosidase was used as thetemplate in a subsequent round of PCR reactions wherein the annealingposition of the 5′-primer was specific to the 5′-terminus of the desiredtruncation and the 3′-primer remained specific for the original3′-terminus of the ORF. To facilitate subcloning of the truncatedmannosidase fragment into the yeast expression vector, pJN347 (FIG. 2C)AscI and PacI restriction sites were engineered onto each truncationproduct, at the 5′- and 3 ′-termini respectively. The number andposition of the N-terminal truncations generated for each mannosidaseORF depended on the position of the transmembrane (TM) region inrelation to the catalytic domain (CD). For instance, if the stem regionlocated between the TM and CD was less than 150 bp, then only onetruncation for that protein was generated. If, however, the stem regionwas longer than 150 bp then either one or two more truncations weregenerated depending on the length of the stem region.

[0292] An example of how truncations for the M. musculus mannosidase IA(Genbank AN 6678787) were generated is described herein, with a similarapproach being used for the other mannosidases. FIG. 3 illustrates theORF of the M. musculus α-1,2-mannosidase IA with the predictedtransmembrane and catalytic domains being highlighted in bold. Based onthis structure, three 5′-primers were designed (annealing positionsunderlined in FIG. 3) to generate the Δ65-, Δ105- and Δ187-N-terminaldeletions. Using the Δ65 N-terminal deletion as an example the 5′-primerused was 5′-GGCGCGCCGACTCCTCCAAGCTGCTCAGCGGGGTCCTGTTCCAC-3′ (SEQ ID NO:14) (with the AscI restriction site highlighted in bold) in conjunctionwith the 3′-primer5′-CCTTAATTAATCATTTCTCTTTGCCATCAATTTCCTTCTTCTGTTCACGG-3′ (SEQ ID NO: 15)(with the PacI restriction site highlighted in bold). Both of theseprimers were used to amplify a 1561 bp fragment under the conditionsoutlined above for amplifying the full length M. musculus mannosidase 1AORF. Furthermore, like the product obtained for the full length ORF, thetruncated product was also incubated with Taq DNA polymerase, ligatedinto pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.), transformed into TOP10and ABI sequenced. After having amplified and confirmed the sequence ofthe truncated mannosidase fragment, the resulting plasmid,pCR2.1-Δ65mMannIA, was digested with AscI and PacI in New EnglandBiolabs buffer #4 (Beverly, Mass.) for 16 h at 37° C. In parallel, thepJN347 (FIG. 2C) was digested with the same enzymes and incubated asdescribed above. Post-digestion, both the pJN347 (FIG. 2C) back-bone andthe truncated catalytic domain were gel extracted and ligated using theQuick Ligation Kit (New England Biolabs, Beverly, Mass.), as recommendedby the manufacturers, and transformed into chemically competent DH5αcells (Invitrogen, Carlsbad, Calif.). Colony PCR was used to confirm thegeneration of the pJN347-mouse Mannosidase IAΔ65 construct.

[0293] Having generated a library of truncated α-1,2-mannosidasecatalytic domains in the yeast expression vector pJN347 (FIG. 2C) theremaining step in generating the targeting peptide/catalytic domainlibrary was to clone in-frame the targeting peptide sequences (FIG. 2).Both the pJN347-mannosidase constructs (FIG. 2D) and thepCR2.1TOPO-targeting peptide constructs (FIG. 2B) such as were incubatedovernight at 37° C. in New England Biolabs buffer #4 in the presence ofthe restriction enzymes NotI and AscI. Following digestion, both thepJN347-mannosidase back-bone and the targeting peptide regions weregel-extracted and ligated using the Quick Ligation Kit (New EnglandBiolabs, Beverly, Mass.), as recommended by the manufacturers, andtransformed into chemically competent DH5α cells (Invitrogen, Carlsbad,Calif.). Subsequently, the pJN347-targeting peptide/mannosidaseconstructs were ABI sequenced to confirm that the generated fusions werein-frame. The estimated size of the final targetingpeptide/alpha-1,2-mannosidase library contains over 1300 constructsgenerated by the approach described above. FIG. 2 illustratesconstruction of the combinatorial DNA library.

[0294] Engineering a P. pastoris OCHI Knock-Out Strain with MultipleAuxotrophic Markers.

[0295] The first step in plasmid construction involved creating a set ofuniversal plasmids containing DNA regions of the KEX1 gene of P.pastoris (Boehm et al. Yeast 1999 May;15(7):563-72) as space holders forthe 5′ and 3′ regions of the genes to be knocked out. The plasmids alsocontained the S. cerevisiae Ura-blaster (Alani et al., Genetics 116,541-545. 1987) as a space holder for the auxotrophic markers, and anexpression cassette with a multiple cloning site for insertion of aforeign gene. A 0.9-kb fragment of the P. pastoris KEX1-5′ region wasamplified by PCR using primersGGCGAGCTCGGCCTACCCGGCCAAGGCTGAGATCATTTGTCCAGCTTCA GA (SEQ ID NO: 16) andGCCCACGTCGACGGATCCGTTTAAACATCGATTGGAGAGGCTGACACC GCTACTA (SEQ ID NO:17)and P. pastoris genomic DNA as a template and cloned into the SacI, SalIsites of pUC19 (New England Biolabs, Beverly, Mass.). The resultingplasmid was cut with BamHI and SalI, and a 0.8-kb fragment of theKEX1-3′ region that had been amplified using primersCGGGATCCACTAGTATTTAAATCATATGTGCGAGTGTACAACTCTTCCC ACATGG (SEQ ID NO:18)and GGACGCGTCGACGGCCTACCCGGCCGTACGAGGAATTTCTCGG ATGACTCTTTTC (SEQ IDNO:19) was cloned into the open sites creating pJN262. This plasmid wascut with BamHI and the 3.8-kb BamHI, BglII fragment of pNKY51 (Alani etal. 1987) was inserted in both possible orientations resulting inplasmids pJN263 (FIG. 4A) and pJN284 (FIG. 4B).

[0296] An expression cassette was created with NotI and PacI as cloningsites. The GAPDH promoter of P. pastoris was amplified using primersCGGGATCCCTCGAGAGATCTTTTTTGTAGAAATGTCTTGGTGCCT (SEQ ID NO:20) andGGACATGCATGCACTAGTGCGGCCGCCACGTGATAGTTGTTCA ATTGATTGAAATAGGGACAA (SEQ IDNO:21) and plasmid pGAPZ-A (Invitrogen) as template and cloned into theBamHI, SphI sites of pUC19 (New England Biolabs, Beverly, Mass.) (FIG.4B). The resulting plasmid was cut with SpeI and SphI and the CYC1transcriptional terminator region (“TT”) that had been amplified usingprimers CCTTGCTAGCTTAATTAACCGCGGCACGTCCGACGGCGGCCCA CGGGTCCCA (SEQ IDNO:22) and GGACATGCATGCGGATCCCTTAAGAGCCGGCAGCTTGCAAATTAAAGCCTTCGAGCGTCCC (SEQ ID NO:23) and plasmid pPICZ-A (Invitrogen) as atemplate was cloned into the open sites creating pJN261 (FIG. 4B).

[0297] A knockout plasmid for the P. pastoris OCHI gene was created bydigesting pJN263 with SalI and SpeI and a 2.9-kb DNA fragment of theOCH1-5′ region, which had been amplified using the primersGAACCACGTCGACGGCCATTGCGGCCAAAACCTTTTTTCCTATT CAAACACAAGGCATTGC (SEQ IDNO:24) and CTCCAATACTAGTCGAAGATTATCTTCTACGGTGCCTGGACTC (SEQ ID NO:25)and P. pastoris genomic DNA as a template, was cloned into the opensites (FIG. 4C). The resulting plasmid was cut with EcoRI and PmeI and a1.0-kb DNA fragment of the OCH1-3′ region that had been generated usingthe primers TGGAAGGTTTAAACAAAGCTAGAGTAAAATAGATATAGCGAG ATTAGAGAATG (SEQID NO:26) and AAGAATTCGGCTGGAAGGCCTTGTACCTTGATGTAGTTCCCGTT TTCATC (SEQID NO:27) was inserted to generate pJN298 (FIG. 4C). To allow for thepossibility to simultaneously use the plasmid to introduce a new gene,the BamHI expression cassette of pJN261 (FIG. 4B) was cloned into theunique BamHI site of pJN298 (FIG. 4C) to create pJN299 (FIG. 4E).

[0298] The P. pastoris Ura3-blaster cassette was constructed using asimilar strategy as described in Lu. P., et al. 1998 (Cloning anddisruption of the P-isopropylmalate dehydrogenase gene (Leu2) of Pichiastipidis with URA3 and recovery of the double auxotroph. Appl.Microbiol. Biotechnol. 49, 141-146.) A 2.0-kb PstI, SpeI fragment of P.pastoris URA3 was inserted into the PstI, XbaI sites of pUC19 (NewEngland Biolabs, Beverly, Mass.) to create pJN306 (FIG. 4D). Then a0.7-kb SacI, PvuII DNA fragment of the lacZ open reading frame wascloned into the SacI, SmaI sites to yield pJN308 (FIG. 4D). Followingdigestion of pJN308 (FIG. 4D) with PstI, and treatment with T4 DNApolymerase, the SacI-PvuII fragment from lacZ that had been blunt-endedwith T4 DNA polymerase was inserted generating pJN315 (FIG. 4D). ThelacZ/URA3 cassette was released by digestion with SacI and SphI, bluntended with T4 DNA polymerase and cloned into the backbone of pJN299 thathad been digested with Pmel and AflII and blunt ended with T4 DNApolymerase. The resulting plasmid was named pJN329 (FIG. 4E).

[0299] A HIS4 marked expression plasmid was created by cutting pJN261(FIG. 4F) with EcoICRI (FIG. 4F). A 2.7 kb fragment of the Pichiapastoris HIS4 gene that had been amplified using the primersGCCCAAGCCGGCCTTAAGGGATCTCCTGATGACTGACTCACTGATAATA AAAATACGG (SEQ IDNO:28) and GGGCGCGTATTTAAATACTAGTGGATCTATCGAATCTAAATGTAAGTTA AAATCTCTAA(SEQ ID NO:29) cut with NgoMIV and SwaI and then blunt-ended using T4DNA polymerase, was then ligated into the open site. This plasmid wasnamed pJN337 (FIG. 4F). To construct a plasmid with a multiple cloningsite suitable for fusion library construction, pJN337 was cut with NotIand PacI and the two oligonucleotidesGGCCGCCTGCAGATTTAAATGAATTCGGCGCGCCTTAAT (SEQ ID NO:30) andTAAGGCGCGCCGAATTCATTTAAATCTGCAGGGC (SEQ ID NO:31), that had beenannealed in vitro were ligated into the open sites, creating pJN347(FIG. 4F).

[0300] To create an och1 knockout strain containing multiple auxotrophicmarkers, 100 μg of pJN329 was digested with Sfil and used to transformP. pastoris strain JC308 (Cereghino et al. Gene 263 (2001) 159-169) byelectroporation. Following transformation, the URA dropout plates wereincubated at room temperature for 10 days. One thousand (1000) colonieswere picked and restreaked. All 1000 clones were then streaked onto 2sets of U-RA dropout plates. One set was incubated at room temperature,whereas the second set was incubated at 37° C. The clones that wereunable to grow at 37° C., but grew at room temperature, were subjectedto colony PCR to test for the correct OCHI knockout. One clone thatshowed the expected PCR signal (about 4.5 kb) was designated YJN153.

EXAMPLE 12 Characterization of the Combinatorial DNA Library

[0301] Positive transformants screened by colony PCR confirmingintegration of the mannosidase construct into the P. pastoris genomewere subsequently grown at room temperature in 50 ml BMGY bufferedmethanol-complex medium consisting of 1% yeast extract, 2% peptone, 100mM potassium phosphate buffer, pH 6.0, 1.34% yeast nitrogen base,4×10⁻⁵% biotin, and 1% glycerol as a growth medium) until OD_(600 nm)2-6 at which point they were washed with 10 ml BMMY (bufferedmethanol-complex medium consisting of 1% yeast extract, 2% peptone, 100mM potassium phosphate buffer, pH 6.0, 1.34% yeast nitrogen base,4×10⁻⁵% biotin, and 1.5% methanol as a growth medium) media prior toinduction of the reporter protein for 24 hours at room temperature in 5ml BMMY. Consequently, the reporter protein was isolated and analyzed bymass spectrophotometry and HPLC to characterize its glycan structure.Using the targeting peptides in Table 6, mannosidase catalytic domainslocalized to either the ER or the Golgi showed significant level oftrimming of a glycan predominantly containing Man₈GlcNAc₂ to a glycanpredominantly containing Man₅GlcNAc₂. This is evident when the glycanstructure of the reporter glycoprotein is compared between that of P.pastoris och1 knock-out in FIGS. 5C, 6C and the same strain transformedwith M. musculus mannosidase constructs as shown in FIGS. 5D, 5E, 6D-6F.FIGS. 5 and 6 show expression of constructs generated from thecombinatorial DNA library which show significant mannosidase activity inP. pastoris. Expression of pGC5 (Saccharomyces MNS1(m)/mouse mannosidaseIB Δ99) (FIGS. 5D, 6E) produced a protein which has approximately 30% ofall glycans trimmed to Man₅GlcNAc₂, while expression of pFB8(Saccharomyces SEC12(m)/mouse mannosidase IA Δ187) (FIG. 6F) producedapproximately 50% Man₅GlcNAc₂ and expression of pBC18-5 (SaccharomycesVAN1(s)/C. elegans mannosidase IB Δ80) (FIG. 5E) produced 70%Man₅GlcNAc₂.

[0302] Release of N-Glycans

[0303] The glycans were released and separated from the glycoproteins bya modification of a previously reported method (Papac et al. 1998Glycobiology 8, 445-454). After the proteins were reduced andcarboxymethylated and the membranes blocked, the wells were washed threetime with water. The protein was deglycosylated by the addition of 30 μlof 10 mM NH4HCO3 pH 8.3 containing one milliunit of N-glycanase (Glyko,Novato, Calif.). After 16 hr at 37° C., the solution containing theglycans was removed by centrifugation and evaporated to dryness.

[0304] Matrix Assisted Laser Desorption Ionization Time of Flight MassSpectrometry

[0305] After the N-glycans were released by PNGase digestion, they wereanalyzed by Matrix Assisted Laser Desorption Ionization Time of FlightMass Spectrometry. Molecular weights of the glycans were determinedusing a Voyager DE PRO linear MALDI-TOF (Applied Biosciences) massspectrometer using delayed extraction. The dried glycans from each wellwere dissolved in 15 μl of water and 0.5 μl was spotted on stainlesssteel sample plates and mixed with 0.5 μl of S-DHB matrix (9 mg/ml ofdihydroxybenzoic acid, 1 mg/ml of 5-methoxysalicilic acid in 1:1water/acetonitrile 0.1% TFA) and allowed to dry. Ions were generated byirradiation with a pulsed nitrogen laser (337 nm) with a 4 ns pulsetime. The instrument was operated in the delayed extraction mode with a125 ns delay and an accelerating voltage of 20 kV. The grid voltage was93.00%, guide wire voltage was 0.1%, the internal pressure was less than5×10-7 torr, and the low mass gate was 875 Da. Spectra were generatedfrom the sum of 100-200 laser pulses and acquired with a 500 MHzdigitizer. Man₅GlcNAc₂ oligosaccharide was used as an external molecularweight standard. All spectra were generated with the instrument in thepositive ion mode.

EXAMPLE 13 Trimming In Vivo by alpha-1,2-mannosidase

[0306] To ensure that the novel engineered strains of Example 11 in factproduced the desired Man₅GlcNAc₂ structure in vivo, cell supernatantswere tested for mannosidase activity (see FIGS. 7-9). For eachconstruct/host strain described below, HPLC was performed at 30° C. witha 4.0 mm×250 mm column of Altech (Avondale, Pa., USA) Econosil-NH₂ resin(5 μm) at a flow rate of 1.0 ml/min for 40 min. In FIGS. 7 and 8,degradation of the standard Man₉GlcNAc₂ [b] was shown to occur resultingin a peak which correlates to Man₈GlcNAc₂. In FIG. 7, the Man₉GlcNAc₂[b] standard eluted at 24.61 min and Man₅GlcNAc₂ [a] eluted at 18.59min. In FIG. 8, Man₉GlcNAc₂ eluted at 21.37 min and Man₅GlcNAc₂ at 15.67min. In FIG. 9, the standard Man₈GlcNAc₂ [b] was shown to elute at 20.88min.

[0307]P. pastoris cells comprising plasmid pFB8 (Saccharomyces SEC12(m)/mouse mannosidase IA Δ187) were grown at 30° C. in BMGY to an OD600of about 10. Cells were harvested by centrifugation and transferred toBMMY to induce the production of K3 (kringle 3 from human plasminogen)under control of an AOX1 promoter. After 24 hours of induction, cellswere removed by centrifugation to yield an essentially clearsupernatant. An aliquot of the supernatant was removed for mannosidaseassays and the remainder was used for the recovery of secreted solubleK3. A single purification step using CM-sepharose chromatography and anelution gradient of 25 mM NaAc, pH5.0 to 25 mM NaAc, pH5.0, 1M NaCl,resulted in a 95% pure K3 eluting between 300-500 mM NaCl. N-glycananalysis of the K3 derived glycans is shown in FIG. 6F. The earlierremoved aliquot of the supernatant was further tested for the presenceof secreted mannosidase activity. A commercially available standard of2-aminobenzamide-labeled N-linked-type oligomannose 9 (Man9-2-AB)(Glyko, Novato, Calif.) was added to: BMMY (FIG. 7A), the supernatantfrom the above aliquot (FIG. 7B), and BMMY containing 10 ng of 75 mU/mLof α-1,2-mannosidase from Trichoderma reesei (obtained from Contreras etal., WO 02/00856 A2) (FIG. 7C). After incubation for 24 hours at roomtemperature, samples were analyzed by amino silica HPLC to determine theextent of mannosidase trimming.

[0308]P. pastoris cells comprising plasmid pGC5 (SaccharomycesMNS1(m)/mouse mannosidase IB Δ99) were similarly grown and assayed.Cells were grown at room temperature in BMGY to an OD600 of about 10.Cells were harvested by centrifugation and transferred to BMMY to inducethe production of K3 under control of an AOX1 promoter. After 24 hoursof induction, cells were removed by centrifugation to yield anessentially clear supernatant. An aliquot of the supernatant was removedfor mannosidase assays and the remainder was used for the recovery ofsecreted soluble K3. A single purification step using CM-sepharosechromatography and an elution gradient of 25 mM NaAc, pH5.0 to 25 mMNaAc, pH5.0, 1M NaCl, resulted in a 95% pure K3 eluting between 300-500mM NaCl. N-glycan analysis of the K3 derived glycans is shown in FIG.5D. The earlier removed aliquot of the supernatant was further testedfor the presence of secreted mannosidase activity as shown in FIG. 8B. Acommercially available standard of Man9-2-AB (Glyko, Novato, Calif.)were added to: BMMY (FIG. 8A), supernatant from the above aliquot (FIG.8B), and BMMY containing 10 ng of 75 mU/mL of α-1,2-mannosidase fromTrichoderma reesei (obtained from Contreras et al., WO 02/00856 A2)(FIG. 8C). After incubation for 24 hours at room temperature, sampleswere analyzed by amino silica HPLC to determine the extent ofmannosidase trimming.

[0309] Man9-2-AB was used as a substrate and it is evident that after 24hours of incubation, mannosidase activity was virtually absent in thesupernatant of the pFB8 (Saccharomyces SEC12 (m)/mouse mannosidase IAΔ187) strain digest (FIG. 7B) and pGC5 (Saccharomyces MNS1(m)/mousemannosidase IB Δ99) strain digest (FIG. 8B) whereas the positive control(purified α-1,2-mannosidase from T reesei obtained from Contreras) leadsto complete conversion of Man₉GlcNAc₂ to Man₅GlcNAc₂ under the sameconditions, as shown in FIG. 7C and 8C. This is conclusive data showingin vivo mannosidase trimming in P. pastoris pGC5 strain; and pFB8strain, which is distinctly different from what has been reported todate (Contreras et al., WO 02/00856 A2).

[0310]FIG. 9 further substantiates localization and activity of themannosidase enzyme. P. pastoris comprising pBC18-5 (SaccharomycesVAN1(s)/C. elegans mannosidase IB Δ80) was grown at room temperature inBMGY to an OD600 of about 10. Cells were harvested by centrifugation andtransferred to BMMY to induce the production of K3 under control of anAOX1 promoter. After 24 hours of induction, cells were removed bycentrifugation to yield an essentially clear supernatant. An aliquot ofthe supernatant was removed for mannosidase assays and the remainder wasused for the recovery of secreted soluble K3. A single purification stepusing CM-sepharose chromatography and an elution gradient 25 mM NaAc,pH5.0 to 25 mM NaAc, pH5.0, 1M NaCl, resulted in a 95% pure K3 elutingbetween 300-500 mM NaCl. N-glycan analysis of the K3 derived glycans isshown in FIG. 5E. The earlier removed aliquot of the supernatant wasfurther tested for the presence of secreted mannosidase activity asshown in FIG. 9B. A commercially available standard of Man8-2-AB (Glyko,Novato, Calif.) was added to: BMMY (FIG. 9A), supernatant from the abovealiquot pBC18-5 (Saccharomyces VAN1(s)/C. elegans mannosidase IB Δ80)(FIG. 9B), and BMMY containing media from a different fusion constructpDD28-3 (Saccharomyces MNN10(m) (from SwissProt 50108)/H. sapiensmannosidase IB Δ99) (FIG. 9C). After incubation for 24 hours at roomtemperature, samples were analyzed by amino silica HPLC to determine theextent of mannosidase trimming. FIG. 9B demonstrates intracellularmannosidase activity in comparison to a fusion construct pDD28-3(Saccharomyces MNN10(m) H. sapiens mannosidase IB Δ99) exhibiting anegative result (FIG. 9C).

EXAMPLE 14 pH Optimum Assay of Engineered α-1,2-mannosidase

[0311]P. pastoris cells comprising plasmid pBB27-2 (Saccharomyces MNN10(s) (from SwissProt 50108)/C. elegans mannosidase IB Δ31) were grown atroom temperature in BMGY to an OD600 of about 17. About 80 μL of thesecells were inoculated into 600 μL BMGY and were grown overnight.Subsequently, cells were harvested by centrifugation and transferred toBMMY to induce the production of K3 (kringle 3 from human plasminogen)under control of an AOX1 promoter. After 24 hours of induction, cellswere removed by centrifuigation to yield an essentially clearsupernatant (pH 6.43). The supernatant was removed for mannosidase pHoptimum assays. Fluorescence-labeled Man₈GlcNAc₂ (0.5 μg) was added to20 μL of supernatant adjusted to various pH (FIG. 11) and incubated for8 hours at room temperature. Following incubation the sample wasanalyzed by HPLC using an Econosil NH2 4.6×250 mm, 5 micron bead,amino-bound silica column (Altech, Avondale, Pa.). The flow rate was 1.0ml/min for 40 min and the column was maintained to 30° C. After elutingisocratically (68% A:32% B) for 3 min, a linear solvent gradient (68%A:32% B to 40% A:60% B) was employed over 27 min to elute the glycans(18). Solvent A (acetonitrile) and solvent B (ammonium formate, 50 mM,pH 4.5. The column was equilibrated with solvent (68% A:32% B) for 20min between runs.

EXAMPLE 15 Engineering of P. pastoris to Produce N-Glycans with theStructure GlcNAcMan₅GlcNAc₂

[0312] GlcNAc Transferase I activity is required for the maturation ofcomplex and hybrid N-glycans (U.S. Pat. No. 5,834,251). Man₅GlcNAc₂ mayonly be trimmed by mannosidase II, a necessary step in the formation ofhuman glycoforms, after the addition of N-acetylglucosamine to theterminal α-1,3 mannose residue of the trimannose stem by GlcNAcTransferase I (Schachter, 1991 Glycobiology 1(5):453-461). Accordingly,a combinatorial DNA library was prepared including DNA fragmentsencoding suitably targeted catalytic domains of GlcNAc Transferase Igenes from C. elegans and Homo sapiens; and localization sequences fromGLS, MNS, SEC, MNN9, VAN1, ANP1, HOC1, MNN10, MNN11, MNT1, KTR1, KTR2,MNN2, MNN5, YUR1, MNN1, and MNN6 from S. cerevisiae and P. pastorisputative α-1,2-mannosyltransferases based on the homology from S.cerevisiae: D2, D9 and J3, which are KTR homologs. Table 10 includes butdoes not limit targeting peptide sequences such as SEC and OCH1, from P.pastoris and K. lactis GnTI, (See Table 6 and Table 10) TABLE 10 Arepresentative combinatorial library of targeting peptide sequences/catalytic domain for UDP-N-Acetylglucosaminyl Transferase I (GnTI)Catalytic Targeting peptide Domain OCHI(s) OCHI(m) OCHI(l) MNN9(s)MNN9(m) Human, PB105 PB106 PB107 PB104 N/A GnTI, Δ38 Human, NB12 NB13NB14 NB15 NB GnTI, Δ86 C.elegans, OA12 OA13 OA14 OA15 OA16 GnTI, Δ88C.elegans, PA12 PA13 PA14 PA15 PA16 GnTI, Δ35 C.elegans, PB12 PB13 PB14PB15 PB16 GnTI, Δ63 X.leavis, QA12 QA13 QA14 QA15 QA16 GnTI, Δ33X.leavis, QB12 QB13 QB14 QB15 QB16 GnTI, Δ103

[0313] Targeting peptide sequences were selected from OCHI in P.pastoris (long, medium and short) (see Example 11) and MNN9 (SwissProtP39107) in S. cerevisiae short, and medium. Catalytic domains wereselected from human GnTI with a 38 and 86 amino acid N-terminaldeletion, C. elegans (gly-12) GnTI with a 35 and 63 amino acid deletionas well as C. elegans (gly-14) GnTI with a 88 amino acid N-terminaldeletion and X. leavis GnTI with a 33 and 103 amino acid N-terminaldeletion, respectively.

[0314] A portion of the gene encoding human N-acetylglucosaminylTransferase I (MGATI, Accession# NM002406), lacking the first 154 bp,was amplified by PCR using oligonucleotides5′-TGGCAGGCGCGCCTCAGTCAGCGCTCTCG-3′ (SEQ ID NO:32) and 5′-AGGTTAATTAAGTGCTAATTCCAGCTAGG-3′ (SEQ ID NO:33) and vector pHG4.5 (ATCC# 79003) astemplate. The resulting PCR product was cloned into pCR2.1-TOPO and thecorrect sequence was confirmed. Following digestion with AscI and PacIthe truncated GnTI was inserted into plasmid pJN346 to create pNA. Afterdigestion of pJN271 with NotI and AscI, the 120 bp insert was ligatedinto pNA to generate an in-frame fusion of the MNN9 transmembrane domainwith the GnTI, creating pNA 15.

[0315] The host organism is a strain of P. pastoris that is deficient inhypermannosylation (e.g. an och1 mutant), provides the substrateUDP-GlcNAc in the Golgi and/or ER (i.e. contains a functional UDP-GlcNActransporter), and provides N-glycans of the structure Man₅GlcNAc₂ in theGolgi and/or ER (e.g. P. pastoris pFB8 (Saccharomyces SEC12 (m)/mousemannosidase IA Δ187) from above). First, P. pastoris pFB8 wastransformed with pPB103 containing the Kluyveromyces lactis MNN2-2 gene(Genbank AN AF 106080) (encoding UDP-GlcNAc transporter) cloned intoBamHI and BglII site of pBLADE-SX plasmid (Cereghino et al. Gene 263(2001) 159-169). Then the aforementioned combinatorial DNA libraryencoding a combination of exogenous or endogenous GnTI/localizationgenes was transformed and colonies were selected and analyzed for thepresence of the GnTI construct by colony PCR. Our transformation andintegration efficiency was generally above 80% and PCR screening can beomitted once robust transformation parameters have been established.

[0316] Protein Purification

[0317] K3 was purified from the medium by Ni-affinity chromatographyutilizing a 96-well format on a Beckman BioMek 2000 laboratory robot.The robotic purification is an adaptation of the protocol provided byNovagen for their HisBind resin. Another screening method may beperformed using a specific terminal GlcNAc binding antibody, or a lectinsuch as the GSII lectin from Griffonia simplificolia, which bindsterminal GlcNAc (EY Laboratories, San Mateo, Calif.). These screens canbe automated by using lectins or antibodies that have been modified withfluorescent labels such as FITC or analyzed by MALDI-TOF.

[0318] Secreted K3 can be purified by Ni-affinity chromatography,quantified and equal amounts of protein can be bound to a high proteinbinding 96-well plate. After blocking with BSA, plates can be probedwith a GSII-FACS lectin and screened for maximum fluorescent response. Apreferred method of detecting the above glycosylated proteins involvesthe screening by MALDI-TOF mass spectrometry following the affinitypurification of secreted K3 from the supernatant of 96-well culturedtransformants. Transformed colonies were picked and grown to an OD600 of10 in a 2 ml, 96-well plate in BMGY at 30° C. Cells were harvested bycentrifugation, washed in BMMY and resuspended in 250 ul of BMMY.Following 24 hours of induction, cells were removed by centrifugation,the supernatant was recovered and K3 was purified from the supernatantby Ni affinity chromatography. The N-glycans were released and analyzedby MALDI-TOF delayed extraction mass spectrometry as described herein.

[0319] In summary, the methods of the invention yield strains of P.pastoris that produce GlcNAcMan₅GlcNAc₂ in high yield, as shown in FIG.10B. At least 60% of the N-glycans are GlcNAcMan₅GlcNAc₂. To date, noreport exists that describes the formation of GlcNAcMan₅GlcNAc₂ onsecreted soluble glycoproteins in any yeast. Results presented hereinshow that addition of the UDP-GlcNAc transporter along with GnTIactivity produces a predominant GlcNAcMan₅GlcNAc₂ structure, which isconfirmed by the peak at 1457 (m/z) (FIG. 10B).

[0320] Construction of Strain PBP-3:

[0321] The P. pastoris strain expressing K3, (Δoch1, arg-, ade-, his-)was transformed successively with the following vectors. First, pFB8(Saccharomyces SEC12 (m)/mouse mannosidase IA Δ187) was transformed inthe P. pastoris strain by electroporation. Second, pPB103 containingKluyveromyces lactis MNN2-2 gene (Genbank AN AF106080) (encodingUDP-GlcNAc transporter) cloned into pBLADE-SX plasmid (Cereghino et al.Gene 263 (2001) 159-169) digested with BamHI and BglII enzymes wastransformed in the P. pastoris strain. Third, pPB104 containingSaccharomyces MNN9(s)/human GnTI Δ38 encoding gene cloned as NotI-PacIfragment into pJN336 was transformed into the P. pastoris strain.

EXAMPLE 16 Engineering K. lactis Cells to Produce N-Glycans with theStructure Man₅GlcNAc₂

[0322] Identification and Disruption of the K. lactis OCHI Gene

[0323] The OCHI gene of the budding yeast S. cerevisiae encodes a1,6-mannosyltransferase that is responsible for the first Golgilocalized mannose addition to the Man₈GlcNAc₂ N-glycan structure onsecreted proteins (Nakayama et al, J Biol Chem.; 268(35):26338-45 (Dec.15, 1993)). This mannose transfer is generally recognized as the keyinitial step in the fungal specific polymannosylation of N-glycanstructures (Nakanishi-Shindo et al, 1993; Nakayama et al, 1992;Morin-Ganet et al, Traffic 1(1):56-68. (January 2000)). Deletion of thisgene in S. cerevisiae results in a significantly shorter N-glycanstructure that does not include this typical polymannosylation or agrowth defect at elevated temperatures (Nakayama et al, EMBO J.;11(7):2511-9 (July 1992)).

[0324] The Och1p sequence from S. cerevisiae was aligned with knownhomologs from Candida albicans (Genbank accession # AAL49987), and P.pastoris (B. K. Choi et al. in prep) along with the Hoc1 proteins of S.cerevisiae (Neiman et al, Genetics, 145(3):637-45 (Mar 1997) and K.lactis (PENDANT EST database) which are related but distinctmannosyltransferases. Regions of high homology that were in common amongOch1p homologs but distinct from the Hoc1p homologs were used to designpairs of degenerate primers that were directed against genomic DNA fromthe K. lactis strain MG1/2 (Bianchi et al, Current Genetics 12, 185-192(1987)). PCR amplification with primers RCD33(CCAGAAGAATTCAATTYTGYCARTGG) (SEQ ID NO:34) and RCD34(CAGTGAAAATACCTGGNCCNGTCCA) (SEQ ID NO:35) resulted in a 302 bp productthat was cloned and sequenced and the predicted translation was shown tohave a high degree of homology to Och1 proteins (>55% to S. cerevisiaeOch1p).

[0325] The 302 bp PCR product was used to probe a Southern blot ofgenomic DNA from K. lactis strain (MG1/2) with high stringency (Sambrooket al, 1989). Hybridization was observed in a pattern consistent with asingle gene indicating that this 302 bp segment corresponds to a portionof the K. lactis genome and K. lactis (KlOCH1) contains a single copy ofthe gene. To clone the entire KlOCHI gene, the Southern blot was used tomap the genomic locus. Accordingly, a 5.2 kb BamHI/PstI fragment wascloned by digesting genomic DNA and ligating those fragments in therange of 5.2 kb into pUC19 (New England Biolabs, Beverly, Mass.) tocreate a K. lactis subgenomic library. This subgenomic library wastransformed into E. coli and several hundred clones were tested bycolony PCR using RCD 33/34. The 5.2 kb clone containing the predictedKlOCHI gene was sequenced and an open reading frame of 1362 bp encodinga predicted protein that is 46.5% identical to the S. cerevisiae OCHIgene. The 5.2 kb sequence was used to make primers for construction ofan och1::KAN^(R) deletion allele using a PCR overlap method (Davidson etal, Microbiology. 148(Pt 8):2607-15. August 2002). This deletion allelewas transformed into two K. lactis strains and G418 resistant coloniesselected. These colonies were screened by both PCR and for temperaturesensitivity to obtain a strain deleted for the OCHI ORF. The results ofthe experiment show strains which reveal a mutant PCR pattern, whichwere characterized by analysis of growth at various temperatures andN-glycan carbohydrate analysis of secreted and cell wall proteinsfollowing PNGase digestion. The och1 mutation conferred a temperaturesensitivity which allowed strains to grow at 30° C. but not at 35° C.FIG. 12A shows a MALDI-TOF analysis of a wild type K. lactis strainproducing N-glycans of Man₈GlcNAc₂ [c] and higher.

[0326] Identification Cloning and Disruption of the K. lactis MNN1 Gene

[0327]S. cerevisiae MNN1 is the structural gene for the Golgiα-1,3-mannosyltransferase. The product of MNN1 is a 762-amino acid typeII membrane protein (Yip et al., Proc Natl Acad Sci USA. 91(7):2723-7.(1994)). Both N-linked and O-linked oligosaccharides isolated from mnn1mutants lack α-1,3-mannose linkages (Raschke et al., J Biol Chem.,248(13):4660-6. (Jul. 10, 1973).

[0328] The Mnn1p sequence from S. cerevisiae was used to search the K.lactis translated genomic sequences (PEDANT). One 405 bp DNA sequenceencoding a putative protein fragment of significant similarity to Mnn1pwas identified. An internal segment of this sequence was subsequentlyPCR amplified with primers KMN1 (TGCCATCTTTTAGGTCCAGGCCCGTTC) (SEQ IDNO:36) and KMN2 (GATCCCACGACGCATCGTATTTCTTTC), (SEQ ID NO:37) and usedto probe a Southern blot of genomic DNA from K. lactis strain (MG1/2).Based on the Southern hybridization data a 4.2 Kb BamHI-PstI fragmentwas cloned by generating a size-selected library as described herein. Asingle clone containing the K. lactis MNN1 gene was identified by wholecolony PCR using primers KMN1 (SEQ ID NO:36) and KMN2 (SEQ ID NO:37) andsequenced. Within this clone a 2241 bp ORF was identified encoding apredicted protein that was 34% identical to the S. cerevisiae MNN1 gene.Primers were designed for construction of a mnn1::NAT^(R) deletionallele using the PCR overlap method (Davidson et al. 2002).

[0329] This disruption allele was transformed into a strain of K. lactisby electroporation and Noursethoicin resistant transformants wereselected and PCR amplified for homologous insertion of the disruptionallele. Strains that reveal a mutant PCR pattern may be subjected toN-glycan carbohydrate analysis of a known reporter gene.

[0330]FIG. 12B depicts the N-glyeans from the K. lactis och1 mnn1deletion strain observed following PNGase digestion the MALDI-TOF asdescribed herein. The predominant peak at 1908 (m/z) indicated as [d] isconsistent with the mass of Man₉GlcNAc₂.

EXAMPLE 17 Engineering Plant Cells To Express GlcNAc Transferases orGalactosyltransferases

[0331] GlcNAc transferase IV is required for the addition of β1,4 GlcNActo the α-1,6 mannose residue and the α-1,3 mannose residues in complexN-glycans in humans. So far GlcNAc transferase IV has not been detectedin or isolated from plants. A transgenic plant that is capable of addinghuman-like N-glycans must therefore be engineered to express GlcNActransferase IV. Thus, the plant host cell or transgenic plant must alsolocalize an expressed GlcNAc transferase IV to the correct intracellularcompartment in the host so that the enzyme can add the β1,4 GlcNAc tothe appropriate mannose residues.

[0332] There is some evidence that glycosyltransferases from mammals andplants have similar targeting signals. For example, a full-length ratα-2,6-sialyltransferase has been shown to correctly localize to thetrans Golgi network in transgenic arabidopsis though not necessarilyactive (Wee E et al. Plant Cell 1998 October; 10(10): 1759-68). A fusionconstruct having fifty-two N-terminal amino acids fromα-2,6-sialyltransferase fused to a green fluorescent reporter protein(GFP) was also shown to correctly localize to the plant Golgi (Boevinket al. Plant J 1998 August; 15(3):441-7). Two mammalian proteins—TGN30and furin—and AtELP, an arabidopsis integral membrane protein(Sanderfoot et al. Proc Natl Acad Sci USA Aug. 18, 1998;95(17):9920-5),which localize to the trans Golgi network, each contain a tyrosinetetrapeptide motif which targets them to the Golgi, probably by arecycling mechanism via the plasma membrane. Although mammals and plantsappear to share some common mechanisms related to protein targeting,exogenous glycosylases may nonetheless not target correctly in a plantcell, however, localization does not necessarily equal enzyme activity.It therefore becomes essential to devise means to correctly target in aplant cell these enzymes and/or other enzymes which participate informing complex, human-like N-glycans.

[0333] Glycosylation enzymes are integral membrane proteins which residein the endoplasmic reticulum and Golgi apparatus. The targeting andlocalization signals are normally contained in the cytoplasmic and/ortransmembrane domains and in some cases are contained in some lumenalamino acids. For example, fifty-two amino acids that make up thetransmembrane domain, nine cytoplasmic amino acids and twenty-sixlumenal amino acids of α-2,6-sialyltransferase are required to targetGFP to the trans Golgi network (Boevink et al. Plant J 1998August;15(3):441-7).

[0334] Thus, a library of sequences encoding cellular targeting signalpeptides comprising of either just the cytoplasmic and transmembranedomains or the cytoplasmic, transmembrane and lumenal domains ofendoplasmic reticulum and Golgi specific proteins is generated, asdescribed in Example 11. The targeting peptide sequences maybe chosenfrom ER and Golgi-resident plant, yeast or animal proteins. Aglycosylation related protein, e.g., an enzyme (or catalytic domainthereof) such as a glycosylase or integral membrane enzyme can be fusedin-frame to the library of targeting peptide sequences and introducedinto plants (FIG. 13). Plant targeting peptide sequences may be mostefficient in localizing the chimeric enzymes to the ER and Golgi,although targeting peptide sequences from fungi and mammals may also beeffective. For example, the N-terminal 77 amino acids from tobaccoN-acetylglucosaminyl Transferase I have been shown to correctly target areporter protein to the Golgi (Ess1 D. et al., FEBS Lett Jun. 18,1999;453(1-2):169-73). In one embodiment, one or more N-terminalfragments comprising these 77 amino acids (or subsets of these aminoacids) is fused to one or more fragments comprising a catalytic domainof GlcNAc transferase IV. At least one resulting fusion proteincorrectly localizes a functional GlcNAc transferase IV to the Golgiapparatus in a plant cell, as evidenced by monitoring the glycosylationstate of a reporter glycoprotein resident or introduced into the planthost cell using techniques described herein.

[0335] Another plant enzyme shown to localize to the Golgi isArabidopsis GlcNAc transferase II (Strasser R et al., Glycoconj J 1999December;16(12):787-91). Thus, in another embodiment, one or moredifferent fragments of the arabidopsis GlcNAc transferase II targetingpeptide are fused to a GlcNAc transferase IV catalytic domain and fusionconstructs produced and tested as described above. The plant specificβ1,2-xylosyltransferase from Arabidopsis thaliana is another proteinthat localizes to the Golgi and its localization and retention in theGolgi is dependent on its cytoplasmic and transmembrane sequences(Dirnberger et al., Plant Mol Biol 2002 September;50(2):273-81). Thus,in another embodiment, one or more fragments comprising the cytoplasmicand transmembrane sequences of β1,2-xylosyltransferase are fused to oneor more fragments comprising a GlcNAc transferase IV catalytic domainand resulting fusion constructs are transformed into plant cells andtested for their ability to produce a human-like N-glycan and tootherwise modulate glycosylation in the plant host cell.

[0336] Because GlcNAc transferase IV or Galactosyltransferase from oneorganism may function more efficiently in a specific plant host than onefrom another organism, fragments comprising GlcNAc transferase IVs (orcatalytic domains) from various eukaryotic organisms are fused in-frameto the library of endoplasmic reticulum (ER) and Golgi targeting peptidesequences and are then introduced into plants. The use of a library ofnucleic acids encoding enzyme domains isolated or derived from differentspecies increases the chances of efficient glycosylation—in addition tocorrect localization and glycosylation by GlcNAc transferase IV.

[0337] The methods and combinatorial nucleic acid libraries of theinvention may be used to introduce and localize, sequentially or enmasse, multiple enzymes required to glycosylate proteins in a plant cellwith human-like N-glycans. As different plant species may requiredifferent growth conditions, protocols for transformation may varydepending on the species being transformed (Potrykus, “Gene transfermethods for plants and cell cultures.” Ciba Found Symp 1990;154:198-208;discussion 208-12). The commonly used methods for generating transgenicplants include Agrobacterium mediated transformation, particlebombardment (Sanford, J. C. et al, Biolistic plant transformation.Physiol. Plant. 1990, 79: 206-209) and electroporation.

[0338] Agrobacterium Method

[0339] The catalytic domains of GlcNAc transferase IVs are fusedin-frame to multiple different targeting peptide sequences known totarget proteins to the ER and Golgi in plants. Each of these fusionconstructs is introduced under the control of the ubiquitously expressedpromoters like the 35S CaMV, ubiquitin or actin promoters, tissuespecific promoters or inducible promoters. A plant specific terminatorregion is also used. This cassette (Promoter::targeting peptide-GlcNActransferase IV::terminator) is cloned into a vector suitable forAgrobacterium mediated transformation (FIG. 13). The vector alsocontains a selectable marker that allows one to select for transformedplants. The common selectable markers used include those resulting inkanamycin, hygromycin and basta resistance. The construct is introducedinto Agrobacterium via well-established transformation methods, whichare available in the art. An Agrobacterium library of Golgi-targetedGlcNAc transferase IVs is thereby generated.

[0340] Embryonic and meristematic tissue may be transformed and canregenerate transgenic plants. To transform tissue, tissue explants(these could be plumules and radicals from germinated seeds) are firstsoaked and coated with an Agrobacterium innoculum. They are thencultured on plates containing the innoculum to form an undifferentiatedmass of cells termed the callus. Transformed plant cells are selectedfor by adding to the medium the relevant kanamycin, hygromycin or basta(depending on the selectable marker used on the construct). Thetransformed plant cells can either be grown in culture and remainundifferentiated or they are treated with shoot regenerating and shootelongation medium. Explants that differentiate are transferred ontorooting medium to generate transgenic plants. Some plants likeArabidopsis can be transformed by dipping flowers into an Agrobacteriumsolution. Seeds from the transformed plants are germinated on platescontaining the relevant herbicide or antibiotic selection. Transgenicplants are those that grow on the selection media. The transgenic plantsare then screened for those with properly glycosylated proteins (i.e.,those which have complex, human-like N-glycans) by isolatingglycoproteins from plant extracts and analyzing glycoprotein patterns asdescribed elsewhere herein, e.g., by using a specific antibody orlectin. Although the Agrobacterium method is economical and simple, itis limited to certain species of plants. Accordingly, plants that cannotbe transformed using Agrobacterium can be transformed by ballistics orelectroporation.

[0341] Particle Bombardment Method and Electroporation

[0342] Compared to Agrobacterium mediated transformation, these methodshave a greater tendency to insert multiple copies of the transgene intothe genome. This could result in gene silencing and cosuppression.However, unlike Agrobacterium mediated transformation, these methods arenot species limited and are therefore useful when an Agrobacteriummethod cannot be employed to generate transgenic plants. In the particlebombardment method, cultured plant cells are bombarded with very smalltungsten or gold particle that have been coated with DNA(Promoter::targeting peptide-GlcNAc transferaseIV-terminator::selectable marker) (FIG. 13) (rb and lb not required)while in the electroporation method, plant cells in a DNA(Promoter::targeting peptide-GlcNAc transferaseIV-terminator::selectable marker) solution are treated with an electricpulse that perforates the cell, allowing it to take up DNA. The cellsare then cultured and allowed to recover. Stable transformants areselected for by culturing and regenerating plants on appropriateselection medium.

[0343] Engineering Soybeans to Express GlcNAc Transferase IV Using aSoybean Cotyledonary Node Agrobacterium Mediated Transformation System

[0344] An Agrobacterium library of Golgi-targeted GlcNAc transferase IVis generated as described above. Soybean explants are transformed withthe library using a protocol described by Hinchee et al (Bio/Technology1988. 6:915). A reporter protein is expressed with a His tag, purifiedand then analyzed. Transgenic plants are assayed for proteins with theα-1,6 mannose and the α-1,3 mannose residues using, e.g., massspectroscopy.

[0345] Engineering Pea to Express GlcNAc Transferase IV Using ParticleBombardment

[0346] A GlcNAc transferase IV plasmid library is coated onto tungstenor gold particles and used as microprojectiles to bombard calli derivedfrom pea embryonic tissue as described (Molnar et al., Symposium onRecent Advances in Plant Biotechnology, Sep. 4-11, 1999, Stara Lesna,Slovak Republic). A reporter protein is expressed with a His tag,purified and then analyzed. Transgenic plants are assayed for proteinswith the α-1,6 mannose and the α-1,3 mannose residues using, e.g.,MALDI.

[0347] Engineering Plants to Express GlcNAc Transferase I

[0348] GlcNAc transferase I is involved in the addition of GlcNAc to theterminal α-1,3 mannose residue to form Man₅GlcNAc₂, an essential step inthe maturation of complex N-glycans. Although GlcNAc transferase I hasbeen isolated from plants and appears to have the same function as itsmammalian homolog, it may not be the most efficient enzyme forglycosylation of mammalian or exogenous proteins and may not be found inevery plant species. As the addition of GlcNAc to the terminal α-1,3mannose residue is a controlling step in the mammalian glycosylationpathway, it is advantageous to have transgenic plants that can carry outthis step efficiently. To create transgenic plants that express GlcNActransferase I that can function efficiently to promote the formation ofcomplex N-glycans, a library of GlcNAc transferase I isolated or derivedfrom various organisms is fused in-frame to multiple plant Golgitargeting peptide sequences according to the methods described herein.The combinatorial library thus created is introduced into a plant cellor organism as described above for GlcNAc transferase IV.

[0349] Engineering Maize to Express GlcNAc Transferase I Using ParticleBombardment

[0350] Transgenic maize can be obtained using a protocol similar to theone used to generate peas that express GlcNAc transferase IV. Here theGlcNAc transferase I plasmid library is coated onto tungsten or goldparticles and used to bombard calli derived from maize embryonic tissue,e.g., using a protocol specific for the generation of transgenic maize(Gordon-Kamm W J et al., Plant Cell 1990 July;2(7):603-618)). Transgenicplants are assayed for proteins having GlcNAc on the terminal α-1,3mannose residue, e.g., using specific antibodies or by assaying reducedbinding of the N-glycans to certain lectins or by using MALDI-TOF.

[0351] Other useful references for using plant host cells according tothe invention include: Christou P. Plant Mol Biol 1997September;35(1-2):197-203; Chowrira G M et al. Mol Biotechnol 1995February;3(1):17-23; Dimberger et al., Plant Mol Biol 2002September;50(2):273-81; Frame B R et al. Plant Physiol 2002May;129(1):13-22; Gomord V et al. Biochimie 1999 June;81(6):607-18;Laursen C M et al. Plant Mol Biol 1994 January;24(1):51-61; Orci L etal. J Cell Biol 2000 Sep. 18;150(6):1263-70; Newell Calif. MolBiotechnol 2000 September; 16(1):53-65; Pawlowski W p et al. MolBiotechnol 1996 August;6(1):17-30; Schroeder H E et al. Plant Physiol1993 March;101(3):751-757; Sorokin, A P et al. Plant Sci. Jul. 28,2000;156(2): 227-233; Strasser R et al. Glycoconj J 1999December;16(12):787-91; and Tomes D T et al. Plant Mol Biol 1990February;14(2):261-8.

[0352] Engineering Plant Cells to Produce β1,4-Galactosyltransferases

[0353] β1,4-galactosyltransferase is an important humanglycosyltransferase that is absent in plants. Lerouge Petal. Plant MolBio 1998 September;38(1-2):31-48. In mammals, β1,4-galactosyltransferaseis localized in the Golgi and is responsible for the transfer ofgalactose residues to the terminal N-acetylglucosamine of the coreMan₃GlcNAc₂ of complex N-glycans. In plants, the Man₃GlcNAc₂ corecontains β1,2-xylose and α1,3-fucose residues and lacks theβ1,4-galactose. The xylose and fucose modifications are implicated inallergies and act as antigenic epitopes and are therefore not desirablemodifications of therapeutic proteins.

[0354] The galactose modifications carried out byβ1,4-galactosyltransferase can be important for the proper functioningof the therapeutic proteins. In mammals, β1,4-galactosyltransferase actsafter N-acetylglucosaminyltransferase I andN-acetylglucosaminyltransferase II and has been shown to initiatebranching of the complex N-glycan. Lerouge P et al. Plant Mol Biol 1998September;38(1-2):31-48. Palacpac N et al. Proc Natl Acad Sci USA Apr.13, 1999;96(8):4692-7. In tobacco cells, expression of humanβ1,4-galactosyltransferase has been shown to result in galactosylatedN-glycans with reduced fucose and xylose modifications. Bakker H et al.Proc Natl Acad Sci USA Feb. 27, 2001;98(5):2899-904 Fujiyama K et al.Biochem Biophys Res Commun Nov. 30, 2001;289(2):553-7. Palacpac N et al.Proc Natl Acad Sci USA Apr. 13, 1999;96(8):4692-7. In these studies, a1.2 kb fragment of human β1,4-galactosyltransferase was cloneddownstream of the cauliflower mosaic virus promoter (35SCaMV),introduced into the binary vector pGA482, and finally into tobaccocells. Palacpac N et al. Proc Natl Acad Sci USA Apr. 13,1999;96(8):4692-7.

[0355] Tobacco cells were transformed using the agrobacterium methoddescribed by Rempel et al. (Rempel, H. C. et al. 1995. Transgenic Res.4(3): 199-207.) Transformation of tobacco cells has also been described(An, G 1985. Plant Physiol. 79:568-570). Expression ofβ1,4-galactosyltransferase under the 35SCaMV resulted in ubiquitousexpression of the gene in tobacco cells. Tobacco cells expressing humanβ1,4-galactosyltransferase showed the presence of galactosylatedN-glycans. (Palacpac N et al. Proc Natl Acad Sci USA Apr. 13,1999;96(8):4692-7). Bakker et al. showed that crossing tobacco plantsexpressing human β1,4-galactosyltransferase with plants expressing theheavy and light chain of a mouse antibody resulted in plants in whichthe antibody showed 30% galactosylation (Bakker H et al. Proc Natl AcadSci USA Feb. 27, 2001;98(5):2899-904).

[0356] A combinatorial DNA library can be constructed to obtain aβ1,4-galactosyltransferase line for the addition of galactose residues.The combinatorial DNA library can effectively produce lines which aremore efficient in the addition of galactose residues. Once such a lineis made it can be easily crossed to lines expressing other glycosylationenzymes and to those expressing therapeutic proteins to producetherapeutic proteins with human-like glycosylation. The final line canthen be grown as plants and harvested to extract proteins or can becultured as plant cells in suspension cultures to produce proteins inbioreactors. By expressing the therapeutic proteins using the library ofsignal peptides, it is possible to retain the therapeutic protein withinthe cells or have them secreted into the medium. Tobacco cellsexpressing β1,4-galactosyltransferase secrete galactosylated N-glycans(Ryo Misaki et al. Glycobiology Dec. 17, 2002; 10:1093). Whilehorseradish peroxidase isozyme C expressed in tobacco plants expressingβ1,4-galactosyltransferase contained xylose and fucose modifications, noxylose or fucose modification could be detected in horseradishperoxidase isozyme C expressed in tobacco cells expressingβ4-galactosyltransferase (GT6 cells). (Fujiyama K et al. Biochem BiophysRes Commun Nov. 30, 2001;289(2):553-7). This indicates that it may beadvantageous to express therapeutic proteins in cell lines instead ofwhole plants.

[0357] Engineering Plants to Produce Sialyltransferase

[0358] In mammals, sialyltransferase is a trans golgi enzyme that addsterminal sialic acid residues to glycosylated polypeptides. Thus far,terminal sialic acid residues have not been detected in plants (Wee E etal. Plant Cell 1998 October;10(10):1759-68). Wee et al. expressed therat α2,6-sialyltransferase in transgenic arabidopsis and showed that theenzyme properly localized to the golgi and was functional. Wee et al.demonstrated that membranes isolated from transgenic arabidopsis, whenincubated with CMP-³H-sialic acid and asialofetuin acceptor, resulted inthe addition of sialic acid residues while membrane isolated fromwild-type arabidopsis did not. While expressing the ratα-2,6-sialyltransferase in arabidopsis resulted in a functional enzymethat was able to incorporate sialic acid residues, fusing the mammalianenzymes α-2,3-sialyltransferase and α-2,6-sialyltransferase to a varietyof transit peptides using the library approach of the present inventiondescribed earlier can result in more efficient sialylation in otherplant species. Wee E et al had to isolate membranes and incubate themwith CMP-³H-sialic acid and asialofetuin acceptor since arabidopsis doesnot have CMP-sialic acid or its transporter. In order to overcome thisadditional step and obtain sialic acid addition in the plant, CMP-sialicacid biosynthetic pathway and the CMP-sialic acid transporter can beco-expressed in transgenic plants expressing α-2,3-sialyltransferase andα-2,6-sialyltransferase. As an alternative the CMP-sialic acidtransporter can be co-expressed α-2,3-sialyltransferase andα-2,6-sialyltransferase in plant cells grown in suspension culture, andCMP-sialic acid or other precursors of CMP-sialic acid supplied in themedium.

[0359] Expressing α-2,3-sialyltransferase and α-2,6-sialyltransferase inLemna

[0360] As described in the U.S. Pat. No. 6,040,498, lemna (duckweed) canbe transformed using both agrobacterium and ballistic methods. Usingprotocols described in the patent, lemna will be transformed with alibrary of golgi targeted α-2,3-sialyltransferase and/orα-2,6-sialyltransferase and a library of mammalian CMP-sialic acidtransporters. Transgenic plants can be assayed for proteins withterminal sialic acid residues.

[0361] Expressing α-2,3-sialyltransferase and α-2,6-sialyltransferase inTobacco Cells

[0362] Alpha-2,3-sialyltransferase and/or α-2,6-sialyltransferase and/ora library of mammalian CMP-sialic acid transporters can also beintroduced into tobacco cells grown in suspension culture as describedfor β1,4-galactosyltransferases. CMP-sialic acid can be added to themedium. Both the cells and the culture medium (secreted proteins) can beassayed for proteins with terminal sialic acid residues residues.

EXAMPLE 18 Engineering Insect Cells to Produce Glycosyltransferases

[0363] Insect cells provide another mechanism for producingglycoproteins but the resulting glycoproteins are not complex human-likeglycoproteins. Marz et al. 1995 Glycoproteins, 29:543-563; Jarvis 1997The Baculoviruses 389-431. It is another feature of the presentinvention to provide enzymes in insect cells, which are targeted to theorganelles in the secretory pathway. In a preferred embodiment, enzymessuch as glycosyltransferases, galactosyltransferases andsialyltransferases are targeted to the ER, Golgi or the trans Golginetwork in lepidopteran insect cells (Sf9). Expression of mammalianβ1,4-galactosyltransferase has been shown in Sf9 cells. Hollister et al.Glycobiology. 1998 8(5):473-480. These enzymes are targeted by means ofa chimeric protein comprising a cellular targeting signal peptide notnormally associated with the enzyme. The chimeric proteins are made byconstructing a nucleic acid library comprising targeting sequences asdescribed herein and the glycosylation enzymes. Baculovirus expressionin insect cells is commonly used for stable transformation for addingmammalian glycosyltransferases in insect cells. Hollister et al.Glycobiology. 2001 11(1):1-9. TABLE 11 DNA and Protein SequenceResources 1. European Bioinformatics Institute (EBI) is a centre forresearch and services in bioinformatics: http: //www.ebi.ac.uk/ 2.Swissprot database: http: //www.expasy.ch/spr 3. List of knownglycosyltransferases and their origin. 4. human cDNA, Kumar et al (1990)Proc. Natl. Acad. Sci. USA 87: 9948-9952 5. human gene, Hull et al(1991) Biochem. Biophys. Res. Commun. 176: 608-615 6. mouse cDNA, Kumaret al (1992) Glycobiology 2: 383-393 7. mouse gene, Pownall et al (1992)Genomics 12: 699-704 8. murine gene (5′ flanking, non-coding), Yang etal (1994) Glycobiology 5: 703-712 9. rabbit cDNA, Sarkar et al (1991)Proc. Natl. Acad. Sci. USA 88: 234-238 10. rat cDNA, Fukada et al (1994)Biosci. Biotechnol. Biochem. 58: 200-201 1,2 (GnTII) EC 2.4.1.143 11.human gene, Tan et al (1995) Eur. J. Biochem. 231: 317-328 12. rat cDNA,D'Agostaro et al (1995) J. Biol. Chem. 270: 15211-15221 13. β1,4(GnTIII) EC 2.4.1.144 14. human cDNA, Ihara et al (1993) J. Biochem.113: 692-698 15. murine gene, Bhaumik et al (1995) Gene 164: 295-300 16.rat cDNA, Nishikawa et al (1992) J. Biol. Chem. 267: 18199-18204 β1,4(GnTIV) EC 2.4.1.145 17. human cDNA, Yoshida et al (1998) GlycoconjugateJournal 15: 1115-1123 18. bovine cDNA, Minowa et al., European Patent EP0 905 232 β1,6 (GnT V) EC 2.4.1.155 19. human cDNA, Saito et al (1994)Biochem. Biophys. Res. Commun. 198: 318-327 20. rat cDNA, Shoreibah etal (1993) J. Biol. Chem. 268: 15381-15385 β1,4 Galactosyltransferase, BC2.4.1.90 (LacNAc synthetase) EC 2.4.1.22 (lactose synthetase) 21. bovinecDNA, D'Agostaro et al (1989) Eur. J. Biochem. 183: 211-217 22. bovinecDNA (partial), Narimatsu et al (1986) Proc. Natl. Acad. Sci. USA 83:4720-4724 23. bovine cDNA (partial), Masibay & Qasba (1989) Proc. Natl.Acad. Sci. USA 86: 5733-5377 24. bovine cDNA (5′ end), Russo et al(1990) J. Biol. Chem. 265: 3324 25. chicken cDNA (partial), Ghosh et al(1992) Biochem. Biophys. Res. Commun. 1215-1222 26. human cDNA, Masri etal (1988) Biochem. Biophys. Res. Commun. 157: 657-663 27. human cDNA,(HeLa cells) Watzele & Berger (1990) Nucl. Acids Res. 18: 7174 28. humancDNA, (partial) Uejima et al (1992) Cancer Res. 52: 6158-6163 29. humancDNA, (carcinoma) Appert et al (1986) Biochem. Biophys. Res. Commun.139: 163-168 30. human gene, Mengle-Gaw et al (1991) Biochem. Biophys.Res. Commun. 176: 1269-1276 31. murine cDNA, Nakazawa et al (1988) J.Biochem. 104: 165-168 32. murine cDNA, Shaper et al (1988) J. Biol.Chem. 263: 10420-10428 33. murine cDNA (novel), Uehara & Muramatsuunpublished 34. murine gene, Hollis et al (1989) Biochem. Biophys. Res.Commun. 162: 1069-1075 35. rat protein (partial), Bendiak et al (1993)Eur. J. Biochem. 216: 405-417 2,3-Sialyltransferase, (ST3Gal II)(N-linked) (Gal-1,3/4-GlcNAc) EC 2.4.99.6 36. human cDNA, Kitagawa &Paulson (1993) Biochem. Biophys. Res. Commun. 194: 375-382 37. rat cDNA,Wen et al (1992) J. Biol. Chem. 267: 21011-21019 2,6-Sialyltransferase.(ST6Gal I) EC 2.4.99.1 38. chicken, Kurosawa et al (1994) Eur. J.Biochem 219: 375-381 39. human cDNA (partial), Lance et al (1989)Biochem. Biophys. Res. Commun. 164: 225-232 40. human cDNA, Grundmann etal (1990) Nucl. Acids Res. 18: 667 41. human cDNA, Zettlmeisl et al(1992) Patent EPO475354-A/3 42. human cDNA, Stamenkovic et al (1990) J.Exp. Med. 172: 641-643 (CD75) 43. human cDNA, Bast et al (1992) J. CellBiol. 116: 423-435 44. human gene (partial), Wang et al (1993) J. Biol.Chem. 268: 4355-4361 45. human gene (5′ flank), Aasheim et al (1993)Eur. J. Biochem. 213: 467-475 46. human gene (promoter), Aas-Eng et al(1995) Biochim. Biophys. Acta 1261: 166-169 47. mouse cDNA, Hamamoto etal (1993) Bioorg. Med. Chem. 1: 141-145 48. rat cDNA, Weinstein et al(1987) J. Biol. Chem. 262: 17735-17743 49. rat cDNA (transcriptfragments), Wang et al (1991) Glycobiology 1: 25-31, Wang et al (1990)J. Biol. Chem. 265: 17849-17853 50. rat cDNA (5′ end), O'Hanlon et al(1989) J. Biol. Chem. 264: 17389-17394; Wang et al (1991) Glycobiology1: 25-31 51. rat gene (promoter), Svensson et al (1990) J. Biol. Chem.265: 20863-20688 52. rat mRNA (fragments), Wen et al (1992) J. Biol.Chem. 267: 2512-2518

[0364] Additional methods and reagents which can be used in the methodsfor modifying the glycosylation are described in the literature, such asU.S. Pat. Nos. 5,955,422, 4,775,622, 6,017,743, 4,925,796, 5,766,910,5,834,251, 5,910,570, 5,849,904, 5,955,347, 5,962,294, 5,135,854,4,935,349, 5,707,828, and 5,047,335. Appropriate yeast expressionsystems can be obtained from sources such as the American Type CultureCollection, Rockville, Md. Vectors are commercially available from avariety of sources.

0 SEQUENCE LISTINGS SEQ ID NO:1-6 can be found in U.S. Pat. applicationNo. 09/892,591 SEQ ID NO:7 Primer: regions of high homology within 1,6mannosyltransferases 5′-atggcgaaggcagatggcagt-3′ SEQ ID NO:8 Primer:regions of high homology within 1,6 mannosyltransferases5′-ttagtccttccaacttccttc-3′ SEQ ID NO:9 internal primer:5′-actgccatctgeettcgccat-3′ SEQ ID NO:10 internal primer:5′-GTAATACGACTCACTATAGGGC-3′ T7 SEQ ID NO:11 Internal primer:5′-AATTAACCCTCACTAAAGGG-3′T3 SEQ ID NO:12 Primer: atgcccgtgg ggggcctgttgccgctcttc agtagc SEQ ID NO:13 Primer: tcatttctct ttgccatcaa tttccttcttctgttcacgg SEQ ID NO:14 Primer: ggcgcgccga ctectccaag ctgctcagcggggtcctgtt ccac SEQ ID NO:15 Primer: ccttaattaatcatttctct ttgccatcaatttccttctt ctgttcacgg SEQ ID NO:16 Primer: ggcgagctcg gcctacccggccaaggctga gatcatttgt ccagcttcaga SEQ ID NO:17 Primer: gcccacgtcgacggatccgt ttaaacatcg attggagagg ctgacaccgc tacta SEQ ID NO:18 Primer:cgggatccac tagtatttaa atcatatgtg cgagtgtaca actettecca catgg SEQ IDNO:19 Primer: ggacgcgtcg acggcctacc cggccgtacg aggaatttct cggatgactcttttc SEQ ID NO:20 Primer: cgggatccct cgagagatct tttttgtaga aatgtcttggtgcct SEQ ID NO:21 Primer: ggacatgcatgcactagtgc ggccgccacg tgatagttgttcaattgatt gaaataggga caa SEQ ID NO:22 Primer: ccttgctagc ttaattaaccgcggcacgtc cgacggcggc ccacgggtcc ca SEQ ID NO:23 Primer: ggacatgcatgcggatccct taagagccgg cagcttgcaa attaaagcct tcgagcgtcc c SEQ ID NO:24Primer: gaaccacgtc gacggccatt gcggccaaaa ccttttttcc tattcaaacacaaggcattg c SEQ ID NO:25 Primer: ctccaatact agtcgaagat tatcttctacggtgcctgga ctc SEQ ID NO:26 Primer: tggaaggtttaaacaaagct agagtaaaatagatatagc gagattagag aatg SEQ ID NO:27 Primer: aagaattcgg ctggaaggccttgtaccttg atgtagttcc cgttttcatc SEQ ID NO:28 Primer: gcccaagccggccttaaggg atctcctgat gactgactca ctgataataa aaatacgg SEQ ID NO:29Primer: gggcgcgta tttaaatacta gtggatctat cgaatctaaa tgtaagttaa aatctctaaSEQ ID NO:30 Primer: ggccgcctgc agatttaaat gaattcgg cgcgccttaat SEQ IDNO:31 Primer: taaggcgcgc cgaattcatt taaatctgca gggc SEQ ID NO:32 Primer:5′-tggcaggcgcgcctcagtcagcgctctcg-3′ SEQ ID NO:33 Primer: 5′-aggttaattaagtgctaattccagctagg-3′ SEQ ID NO:34 primer for K. lactis OCH1 gene:ccagaagaat tcaattytgy cartgg SEQ ID NO:35 primer for K. lactis OCH1gene: cagtgaaaat acctggnccn gtcca SEQ ID NO:36 primer for K. lactis MNN1gene: tgccatcttt taggtccagg cccgttc SEQ ID NO:37 primer for K. lactisMNN1 gene: gatcccacga cgcatcgtat ttctttc SEQ ID NO:38 DNA sequence ofthe 302 bp segment of the putative KlOCH1 gene:gcccttcagtgaaaatacctggcccggtccagttcataatatcggtaccatctgtatttttggcggttttcttttgttgatgtttgtaatttttgttgaacttctttttatccctcatgttgacattataatcatctgcaatgtcttttaatacttcagcatcatctaaaggaatgctgcttttaacatttgccacgctctccaatgttgttgcggtgatatttgtgatcaattcgcgcaataatggatggccagattttgattgtattgtccactgacaaaattgaattctctggaagggc SEQ ID NO:39Translation of putative KlOCH1 gene (excluding primers):TIQSKSGHPLLRELITNITATTLESVANVKSSIPLDDAEVLKDIADDYNVNMRDKKKFNKNYKHQQKKTAKNTDGTDIMN SEQ ID NO:40 DNA sequence of the 405 bpsegment of the putative KlMNN1 gene:cccagcgtgccattaccgtatttgccgccgtttgaaatactcaatattcatgatggttgtaaggcgttttttatcattcgcgatataatatgccatcttttaggtccaggcccgttctcttagctatctttggtgtctgtgctaccgtgatatggtacctattctttttccagtctaatctgaagatggcagatttgaaaaaggtagcaacttcaaggtatctttcacaagaaccgtcgttatcagaacttatgtcaaatgtgaagatcaagcctattgaagaaaccccggtttcgccattggagttgattccagatatcgaaatatcgactagaaagaaatacgatgcgtcgtgggatctgttgttccgtggtagaaaatataaatcgttcaacgattatgatSEQ ID NO:41 DNA sequence of the K. lactis OCH1 gene:atggggttaccaaagatttcaagaagaacgaggtacattattgtcattgtgctgatactgtacttattgttttctgtgcaatggaatactgcgaaagtgaatcaccatttctataacagcattggcacggtgcttcccagtacagctcgcgtggatcacttgaacttgaaaaacttggacttagcaggtacgagcaataacggtgatcatttgatggatctacgagttcaattggctagtcaattcccctacgattctcgagtacccatccccaaaaaggtatggcagacctggaagattgatcccagttcaaagtcacaggtttcttccatttcaaaatgccagaatgattggaaacatttcagtgcatccgaggaaccgccatatcaataccaattaatcacagatgatcaaatgataccacttctagagcagctatatggtggggtcccacaagtgataaaggcttttgaatccttgccacttccaattcttaaagcagactttttcagatacttgatcctttatgcaagaggtggtatatattctgacatggatacgttcccattaaagccattgtcgtcatggccatcgacttctcagtcctacttttctagtttaaagaatccacaaaggtatagaaattccttggacaaccttgaaacgctagaagcttcagaacctggctttgtcattggtatcgaggctgatccggatagaagcgattgggcagagtggtacgccaggagaatacaattctgtcagtggacaatacaatcaaaatctggccatccattattgcgcgaattgatcacaaatatcaccgcaacaacattggagagcgtggcaaatgttaaaagcagcattcctttagatgatgctgaagtattaaaagacattgcagatgattataatgtcaacatgagggataaaaagaagttcaacaaaaattacaaacatcaacaaaagaaaaccgccaaaaatacagatggtaccgatattatgaactggactggtccaggtattttttcagatgttattttccagtatcttaataacgttatccagaagaatgatgacattttaattttcaatgataatcttaatgttatcaacaaacatggatccaaacatgatacaactatgagattctataaagacattgttaaaaatttacaaaacgacaaaccctcattgttctggggattcttttcattgatgacagagcctattctagtggacgacatcatggtacttccgattacttctttctcaccaggtatcagaacaatgggcgctaaagaagacaacgacgagatggcatttgttaagcatatttttgaaggaagttggaaagactga SEQ ID NO:42Translation of putative K. lactis OCH1 gene:MGLPKISRRTRYIIVIVLILYLLFSVQWNTAKVNHHFYNSIGTVLPSTARVDHLNLKNLDLAGTSNNGDHLMDLRVQLASQFPYDSRVPIPKKVWQTWKIDPSSKSQVSSISKCQNDWKHFSASEEPPYQYQLITDDQMIPLLEQLYGGVPQVIKAFESLPLPILKADFFRYLILYARGGIYSDMDTFPLKPLSSWPSTSQSYFSSLKNPQRYRNSLDNLETLEASEPGFVIGIEADPDRSDWAEWYARRIQFCQWTIQSKSGHPLLRELITNITATTLESVANVKSSIPLDDAEVLKDIADDYNVNMRDKKKFNKNYKHQQKKTAKNTDGTDIMNWTGPGIFSDVIFQYLNNVIQKNDDILIFNDNLNVINKHGSKHDTTMRFYKDIVKNLQNDKPSLFWGFFSLMTEPILVDDIMVLPITSFSPGIRTMGAKEDNDEMAFVKHIFEGSWKDZ SEQ ID NO:43 DNA sequenceof the K. lactis MNN1 gene:atgatggttgtaaggcgttttttatcagcttcgcgatataatatgccatcttttaggtccaggcccgttctcttagctatctttggtgtctgtgctaccgtgatatggtacctattctttttccagtctaatctgaagatggcagatttgaaaaaggtagcaacttcaaggtatctttcacaagaaccgtcgttatcagaacttatgtcaaatgtgaagatcaagcctattgaagaaaccccggtttcgccattggagttgattccagatatcgaaatatcgactagaaagaaatacgatgcgtcgtgggatctgttgttccgtggtagaaaatataaatcgttcaacgattatgatcttcatacgaaatgtgagttttatttccagaatttatacaatttgaacgaggattggaccaataatattcggacgttcactttcgatattaacgatgtagacacgtctacgaaaattgacgctcttaaagattccgatggggttcaattggtggacgagaaggctatacgtttatacaagagaacgcataacgttgccttggctacggaaaggttacgtctttatgataaatgttttgtcaatagtccaggttcaaacccattgaaaatggatcaccttttcagatcgaacaagaagagtaagactacggctttggatgacgaagtcactgggaaccgtgatacttttaccaagacgaagaaaacttcgttcttaagcgatatggacacgagtagtttccagaagtacgatcaatgggatttcgaacatagaatgttccccatgatcccatatttcgaggaacacaatttcaccaacgtgatgcctattttcaccggctcaaacggtggggaacctttacctcaagggaaattcccggtattagatccaaaatccggtgaattgttacgtgtagagactttcagatatgataaatcgaaatcgctttggaagaactggaatgatatgtcctctgcttctggtaaacgtggtattatcttggctgctggcgacggccaagtggaccaatgcatccgtcttattgctacgttgagagctcaaggaaacgctctacctattcaaattatccacaacaaccaattgaatgagaaatctgtgaaactgttatcggaggccgctaaatctaccgaattctcatccggtagagctcaatctctttggttagtgaatgtgggccccacgttggaatcttcaatgaagagcaattttgggagatttaagaataagtggttgtcagttattttcaacacttttgaagaatttatattcatagatacagatgccatctcctacattaatatggctgattatttcaacttcaaggagtacaaatctactggaacactcttctttaaggataggtctttggcaattggaactgaacagaaatgtggtcctttgttcgaaactcttgaaccaagaattcttgaaatgtactatttcaatactttacctatgatcaatggtgattacgtggaacagcaatgtatgggcatgctcaccccagaggaaaaagtttacaaacgtttctttgaagttggtcatcaacacaacttggaaagtggattattggccatcaacaaaaacgaacacatcatgggattggttactgcaacagtcttaaatatcgcaccaaaggtcggaggttgcggttggggtgacaaagagtttttctggcttggtttgttggttgctggccaacgctactcgatctatgatatagatgcaagtgcaattggtgttcctcaacagaagcaatctatcgctaacggagacgaatttgatgaatataggatttgttctttacaagtggcacatacttcatacgacggacatttactatggataaatggtggctctcagtactgtaagaaaccagagacttttgaaggtgattggaccaacattaaggagcttcgtgaatcgtattctgatgataaagaaaaggctctgaaggcttatagtgatacagttaaggtggaagcagcaatcgtgccagattccagaagtaatggttggggtagagacgatcaaagatgtaaaggctacttctggtgcggcaaatttacttcaaagctgaaaccgtatacttataacacggtggtaactaaaggtgatttgatccgtttcggagacgaggaaatcgaaagtatctccaagattaataagatctggaatgatgctattattccagacggagcttaa SEQ ID NO:44Translation of putative K. lactis MNN1 gene:MMVVRRFLSASRYNMPSFRSRPVLLAIFGVCATVIWYLFFFQSNLKMADLKKVATSRYLSQEPSLSELMSNVKIKPIEETPVSPLELIPDIEISTRKKYDASWDLLFRGRKYKSFNDYDLHTKCEFYFQNLYNLNEDWTNNIRTFTFDINDVDTSTKIDALKDSDGVQLVDEKAIRLYKRTHNVALATERLRLYDKCFVNSPGSNPLKMDHLFRSNKKSKTTALDDEVTGNRDTFTKTKKTSFLSDMDTSSFQKYDQWDFEHRMFPMIPYFEEHNFTNVMPIFTGSNGGEPLPQGKFPVLDPKSGELLRVETFRYDKSKSLWKNWNDMSSASGKRGIILAAGDGQVDQCIRLIATLRAQGNALPIQIIHNNQLNEKSVKLLSEAAKSTEFSSGRAQSLWLVNVGPTLESSMKSNFGRFKNKWLSVIFNTFEEFIFIDTDAISYINMADYFNFKEYKSTGTLFFKDRSLAIGTEQKCGPLFETLEPRILEMYYFNTLPMINGDYVEQQCMGMLTPEEKVYKRFFEVGHQHNLESGLLAINKNEHIMGLVTATVLNIAPKVGGCGWGDKEFFWLGLLVAGQRYSIYDIDASAIGVPQQKQSIANGDEFDEYRICSLQVAHTSYDGHLLWINGGSQYCKICPETFEGDWTNIKELRESYSDDKEKALKAYSDTVKVEAAIVPDSRSNGWGRDDQRCKGYFWCGKFTSKLKPYTYNTVVTKGDLIRFGDEEIESISKINKIWNDAIIPDGA

What is claimed is:
 1. A method for producing a human-like glycoproteinin a non-human eukaryotic host cell that does not display a 1,6mannosyltransferase activity with respect to the N-glycan on aglycoprotein, the method comprising the step of introducing into thehost cell one or more enzymes for production of a Man₅GlcNAc₂carbohydrate structure, wherein Man₅GlcNAc₂ is produced within the hostcell at a yield of at least 30 mole percent.
 2. The method of claim 1,wherein at least 10 percent of the Man₅GlcNAc₂ produced within the hostcell is a productive substrate for GnTI in vivo.
 3. The method of claim1, wherein at least one of the enzymes is selected to have optimalactivity at the pH of the location in the host cell where thecarbohydrate structure is produced.
 4. The method of claim 2, wherein atleast one of the enzymes is selected to have a pH optimum within about1.4 pH units of the average pH optimum of other representative enzymesin the organelle in which the enzyme is localized.
 5. The method ofclaim 1, wherein at least one of the enzymes is targeted to asubcellular location in the host cell where the enzyme will have optimalactivity.
 6. The method of claim 4, wherein the enzyme is targeted bymeans of a chimeric protein comprising a cellular targeting signalpeptide not normally associated with the enzyme.
 7. The method of claim1, wherein at least one introduced enzyme is targeted to the endoplasmicreticulum, the early, medial, late Golgi or the trans Golgi network ofthe host cell.
 8. The method of claim 1, wherein at least one of theenzymes is selected from the group consisting of mannosidases,glycosyltransferases and glycosidases.
 9. The method of claim 6, whereinthe enzyme is a mannosidase predominantly localized in the Golgiapparatus or the endoplasmic reticulum.
 10. The method of claim 1,wherein the glycoprotein comprises N-glycans of which greater than 30mole percent comprise six or fewer mannose residues.
 11. The method ofclaim 1, wherein the glycoprotein comprises N-glycans of which greaterthan 30 mole percent comprise three or fewer mannose residues.
 12. Themethod of claim 1, wherein the glycoprotein comprises one or more sugarsselected from the group consisting of GlcNAc, galactose, sialic acid,and fucose.
 13. The method of claim 1, wherein the glycoproteincomprises at least one oligosaccharide branch comprising the structureNeuNAc-Gal-GlcNAc-Man.
 14. The method of claim 1, wherein the host isselected from the group consisting of plant, algae, insect, fungi, yeastcells.
 15. The method of claim 1, wherein the host is a lower eukaryoticcell.
 16. The method of claim 15, wherein the host cell is selected fromthe group consisting of Pichia pastoris, Pichia finlandica, Pichiatrehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae,Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichiapijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomycescerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp.,Kluyveromyces lactis, Candida albicans, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporiumlucknowense, Fusarium sp., Fusarium gramineum, Fusarium. venenatum andNeurospora crassa.
 17. The method of claim 1, wherein the host isdeficient in the activity of one or more enzymes selected from the groupconsisting of mannosyltransferases and phosphomannosyltransferases. 18.The method of claim 17, wherein the host does not express an enzymeselected from the group consisting of 1,6 mannosyltransferase; 1,3mannosyltransferase; and 1,2 mannosyltransferase.
 19. The method ofclaim 1, wherein the host is an och1 mutant of P. pastoris.
 20. Themethod of claim 1, wherein the host expresses GnTI and UDP-GlcNActransporter activities.
 21. The method of claim 1, wherein the hostexpresses a UDP- or GDP-specific diphosphatase activity.
 22. The methodof claim 1, further comprising the step of isolating the glycoproteinfrom the host.
 23. The method of claim 22, further comprising the stepof subjecting the isolated glycoprotein to at least one furtherglycosylation reaction in vitro, subsequent to its isolation from thehost.
 24. The method of claim 1, wherein the step of introducing intothe host cell one or more enzymes for production of the Man₅GlcNAc₂carbohydrate structure comprises a nucleic acid molecule.
 25. The methodof claim 1, further comprising the step of introducing into the host anucleic acid molecule encoding one or more mannosidase activitiesinvolved in the production of Man₅GlcNAc₂ from Man₈GlcNAc₂ orMan₉GlcNAc₂.
 26. The method of claim 25, wherein at least one of theencoded mannosidase activities has a pH optimum within about 1.4 pHunits of the average pH optimum of other representative enzymes in theorganelle in which the mannosidase activity is localized, or has optimalactivity at a pH of between about 5.1 and about 8.0.
 27. The method ofclaim 26, wherein the mannosidase has optimal activity at a pH ofbetween about 5.5 and about 7.5.
 28. The method of claim 26, wherein themannosidase activity is an α-1,2-mannosidase derived from mouse, human,Lepidoptera, Aspergillus nidulans, or Bacillus sp., C. elegans, D.melanogaster, P. citrinum or X. laevis.
 29. The method of claim 24,wherein at least one enzyme is localized by forming a fusion proteincomprising a catalytic domain of the enzyme and a cellular targetingsignal peptide.
 30. The method of claim 29, wherein the fusion proteinis encoded by at least one genetic construct formed by the in-frameligation of a DNA fragment encoding a cellular targeting signal peptidewith a DNA fragment encoding a catalytic domain having enzymaticactivity.
 31. The method of claim 30, wherein the encoded targetingsignal peptide is derived from a member of the group consisting of:membrane-bound proteins of the ER or Golgi, retrieval signals, Type IImembrane proteins, Type I membrane proteins, membrane spanningnucleotide sugar transporters, mannosidases, sialyltransferases,glucosidases, mannosyltransferases and phospho-mannosyltransferases. 32.The method of claim 24, wherein the catalytic domain encodes aglycosidase, mannosidase or a glycosyltransferase activity derived froma member of the group consisting of GnTI, GnTII, GnTIII, GnTIV, GnT V,GnT VI, GalT, Fucosyltransferase and Sialyltransferase, and wherein thecatalytic domain has a pH optimum within 1.4 pH units of the average pHoptimum of other representative enzymes in the organelle in which theenzyme is localized, or has optimal activity at a pH between 5.1 and8.0.
 33. The method of claim 32, wherein the catalytic domain encodes amannosidase selected from the group consisting of C. elegans mannosidaseIA, C. elegans mannosidase IB, D. melanogaster mannosidase IA, H.sapiens mannosidase IB, P. citrinum mannosidase I, mouse mannosidase IA,mouse mannosidase IB, A. nidulans mannosidase IA, A. nidulansmannosidase IB, A. nidulans mannosidase IC, mouse mannosidase II, C.elegans mannosidase II, H. sapiens mannosidase II, and mannosidase III.34. The method of claim 24, wherein the nucleic acid molecule encodesone or more enzymes selected from the group consisting of UDP-GlcNActransferase, UDP-galactosyltransferase, GDP-fucosyltransferase,CMP-sialyltransferase, UDP-GlcNAc transporter, UDP-galactosetransporter, GDP-fucose transporter, CMP-sialic acid transporter, andnucleotide diphosphatases.
 35. The method of claim 24, wherein the hostexpresses GnTI and UDP-GlcNAc transporter activities.
 36. The method ofclaim 24, wherein the host expresses a UDP- or GDP-specificdiphosphatase activity.
 37. A nucleic acid library comprising at leasttwo different genetic constructs, wherein at least one genetic constructcomprises a nucleic acid fragment encoding a glycosylation enzymeligated in-frame with a nucleic acid fragment encoding a cellulartargeting signal peptide which it is not normally associated with.
 38. ADNA library of fusion constructs comprising: (a) at least two nucleotidesequences encoding a cellular targeting signal peptide and at least onenucleotide sequence encoding a catalytic domain region selected from thegroup consisting of mannosidases, glycosyltransferases and glycosidases;or (b) at least one nucleotide sequence encoding a cellular targetingsignal peptide and at least two nucleotide sequences encoding acatalytic domain region selected from the group consisting ofmannosidases, glycosyltransferases and glycosidases; wherein at leastone nucleotide sequence encoding a catalytic domain region is ligatedin-frame to a nucleotide sequence encoding a cellular targeting signalpeptide.
 39. The DNA library of claim 37 comprising at least one nucleicacid comprising a naturally occurring sequence encoding a glycosylationenzyme.
 40. The DNA library of claim 37 comprising at least one nucleicacid sequence previously subjected to a technique selected from thelist: gene shuffling, in vitro mutagenesis, and error-prone polymerasechain reaction.
 41. The DNA library of claim 37, wherein theglycosyltransferase is selected from the group consisting of:mannosyltransferases, GlcNAc transferases, phospho-GlcNAc transferases,galactosyltransferases, sialyltransferases and fucosyltransferases. 42.The DNA library of claim 37, wherein at least one nucleotide sequenceencoding a catalytic domain region is derived from mouse, human, C.elegans, D. melanogaster, P. citrinum, X. laevis, Bacillus sp. or A.nidulans.
 43. The DNA library of claim 37, wherein the mannosidasecatalytic domain is selected from the group consisting of: C. elegansmannosidase IA, C. elegans mannosidase IB, D. melanogaster mannosidaseIA, H. sapiens mannosidase IB, P. citrinum mannosidase I, mousemannosidase IA, mouse mannosidase IB, A. nidulans mannosidase IA, A.nidulans mannosidase IB, A. nidulans mannosidase IC, mouse mannosidaseII, C. elegans mannosidase II, H. sapiens mannosidase II, andmannosidase III.
 44. The DNA library of claim 37, wherein the nucleicacid fragment encoding a cellular targeting signal peptide is selectedfrom the group consisting of: membrane-bound proteins of the ER orGolgi, retrieval signals, Type II membrane proteins, Type I membraneproteins, membrane spanning nucleotide sugar transporters, mannosidases,sialyltransferases, glucosidases, mannosyltransferases andphosphomannosyltransferases.
 45. The DNA library of claim 37, whereinthe nucleic acid fragment encoding a cellular targeting peptide isselected from the group consisting of: Saccharomyces GLS1, SaccharomycesMNS1, Saccharomyces SEC12, Pichia SEC, Pichia OCH1, Saccharomyces MNN9,Saccharomyces VAN1, Saccharomyces ANP1, Saccharomyces HOC1,Saccharomyces MNN10, Saccharomyces MNN11, Saccharomyces MNT1, Pichia D2,Pichia D9, Pichia J3, Saccharomyces KTR1, Saccharomyces KTR2,Kluyveromyces GnTI, Saccharomyces MNN2, Saccharomyces MNN5,Saccharomyces YUR1, Saccharomyces MNN1, and Saccharomyces MNN6.
 46. Avector comprising a fusion construct derived from a DNA library of anyone of claims 37-45 operably linked to an expression control sequence,wherein said cellular targeting signal peptide is targeted to the ER,Golgi or trans-Golgi network.
 47. The vector of claim 46 which, uponexpression in a host cell, encodes a mannosidase activity involved inproducing Man₅GlcNAc₂ in vivo.
 48. The vector of claim 46 which, uponexpression in a host cell, encodes a glycosyltransferase activityinvolved in producing GlcNAcMan₅GlcNAc₂ in vivo.
 49. A eukaryotic hostcell comprising at least one vector of claim
 46. 50. The host cell ofclaim 49, selected from the group consisting of unicellular andmulticellular fungi.
 51. The host cell of claim 49, selected from thegroup consisting of Pichia pastoris, Pichia finlandica, Pichiatrehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae,Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichiapijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomycescerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp.,Kluyveromyces lactis, Candida albicans, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporiumlucknowense, Fusarium sp. Fusarium gramineum, Fusarium venenatum andNeurospora crassa.
 52. A method for producing a human-like glycoproteinin a non-human cell comprising the step of culturing a eukaryotic hostcell comprising at least one vector of claim
 46. 53. The method of claim52, wherein the host is a unicellular or multicellular fungal cell. 54.The method of claim 52, wherein the host cell is selected from the groupconsisting of Pichia pastoris, Pichia finlandica, Pichia trehalophila,Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichiathermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi,Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomycescerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp.,Kluyveromyces lactis Candida albicans, Aspergillus nidulans, Aspergillusniger, Aspergillus oryzae, Trichoderma reesei, Chrysosporiumlucknowense, Fusarium sp. Fusarium gramineum, Fusarium venenatum andNeurospora crassa.
 55. A method for producing a human-like glycoproteinin a non-human host cell comprising the step of transforming the hostcell with a DNA library of any one of claims 37-45 to produce agenetically mixed cell population expressing at least one glycosylationenzyme derived from the library.
 56. The method of claim 55, furthercomprising the step of selecting from the mixed cell population a cellproducing a desired human-like glycosylation phenotype.
 57. The methodof claim 56, wherein the selection comprises the step of analyzing aglycosylated protein or isolated N-glycan by one or more methodsselected from the group consisting of: (a) mass spectroscopy; (b)MALDI-TOF; (c) liquid chromatography; (d) characterizing cells using afluorescence activated cell sorter, spectrophotometer, fluorimeter, orscintillation counter; (e) exposing host cells to a lectin or antibodyhaving a specific affinity for a desired oligosaccharide moiety; and (f)exposing cells to a cytotoxic or radioactive molecule selected from thegroup consisting of sugars, antibodies and lectins.
 58. The method ofclaim 55, wherein the DNA fragment encoding the catalytic domain has anactivity selected from the group consisting of mannosidase, UDP-GlcNActransferase, UDP-galactosyltransferase, and CMP-sialyltransferaseactivity and wherein the cellular targeting signal peptide localizes theenzyme predominantly in a host cell organelle selected from the groupconsisting of endoplasmic reticulum, cis Golgi, medial Golgi, and transGolgi.
 59. The method of claim 55, wherein said host cell furthercomprises a target glycoprotein of interest on which Man₅GlcNAc₂ isproduced in vivo.
 60. The method of claim 59, wherein the Man₅GlcNAc₂produced in vivo is the predominant N-glycan on the target glycoprotein.61. The method of claim 55, wherein said host cell further comprises atarget glycoprotein of interest on which GlcNAcMan₅GlcNAc₂ is producedin vivo.
 62. The method of claim 61, wherein the GlcNAcMan₅GlcNAc₂produced in vivo is the predominant N-glycan on the target glycoprotein.63. A host cell produced by the method of claim 1, 24 or
 55. 64. Ahuman-like glycoprotein produced by the method of claim 1, 24 or
 55. 65.A method for altering the glycosylation pattern of a eukaryotic cellcomprising the step of transforming the host cell with a DNA library ofany one of claims 37-45 to produce a genetically mixed cell populationexpressing at least one glycosylation enzyme derived from the library.66. An isolated nucleic acid molecule comprising or consisting ofnucleic acid sequences selected from the group consisting of: (a) atleast forty-five (45) contiguous nucleotide residues of SEQ ID NO:41;(b) homologs, variants and derivatives of (a); and (c) nucleic acidsequences that hybridize under stringent conditions to (a) but excludingsequences which encode the S. cerevisiae OCHI gene.
 67. An isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO:42.
 68. Anisolated nucleic acid molecule of claim 66 which encodes an OCHIactivity upon expression in a host cell.
 69. An isolated nucleic acidmolecule of claim 66 which encodes a K. lactis OCHI gene.
 70. Anisolated nucleic acid molecule comprising or consisting of nucleic acidsequences selected from the group consisting of: (a) at least forty-five(45) contiguous nucleotide residues of SEQ ID NO:43; (b) homologs,variants and derivatives of (a); and (c) nucleic acid sequences thathybridize under stringent conditions to (a) but excluding sequenceswhich encode the S. cerevisiae MNN1 gene.
 71. An isolated polypeptidecomprising the amino acid sequence of SEQ ID NO:44.
 72. An isolatednucleic acid molecule of claim 70 which encodes an MNN1 gene.
 73. Anisolated nucleic acid molecule of claim 70, wherein said sequenceencodes a K. lactis MNN1 gene.
 74. A host cell comprising a disruptionor mutation of SEQ ID NO:41 which is characterized by having a reducedexpression level of SEQ ID NO:41 compared to a host cell without saiddisruption or mutation.
 75. A host cell comprising a disruption ormutation of SEQ ID NO:43 which is characterized by having a reducedexpression level of SEQ ID NO:43 compared to a host cell without saiddisruption or mutation.
 76. A method of modifying plant glycosylationcomprising introducing into a host at least one nucleotide sequenceencoding a catalytic domain region ligated in-frame to a nucleotidesequence encoding a cellular targeting signal peptide.